Biochemistry and Molecular Biology of Parasites

Preface

Parasitology developed as a part of medicine practised in the tropical and subtropical areas of the globe. Later, it also became a major veterinary discipline. Although it has remained in these roles, it has taken on the characteristics of a science in its own right. The organisms which comprise this discipline are varied in their morphologies and astoundingly complex in their life cycles. In the course of these lives they enter and leave ecosystems and thereby undergo significant metabolic and genetic alterations. For these reasons parasitic organisms have become model systems for the study of biochemistry and molecular biology. The phenomena of aerobic fermentation, compartmentalization of enzyme systems, metabolic shifts to accompany morphologic changes, rapid alterations in membrane chemistry, and genetic changes associated with adaptation to the host have recommended parasites to the attention of scientists of many disciplines. The objective of this volume is to present the modern scientific disciplines which have parasitology as a common resource in a setting that will allow and encourage the reader to place the biochemistry and molecular biology of these organisms in their biological context. The format is of a multiauthor volume, in keeping with the many branches of science which have made their places in parasitology. The traditional separations within these have been eliminated in so far as possible to facilitate correlation. The chapters are cross-referenced and grouped in a manner which should be self-reinforcing. For this reason, for example, helminth intermediary metabolism is placed with that of protozoa in order to draw the appropriate parallels and contrasts, rather than placing it in a section devoted to helminths. We believe that this organizational arrangement will permit the reader to create a composite of the biochemistry of all these organisms and not be distracted by their morphological and taxonomic differences. We believe these correlations are important since this discipline has traditionally been taught by artificially subdividing it according to the number of cells in the organism, the residence of the parasite within the intestinal lumen or tissues of the host, or morphology. Science has demonstrated the fundamental unity of biochemistry and molecular biology and parasitology is ripe for unifying concepts. The volume begins with molecular biology which is presented as it relates to the cell biology of these organisms. Although protozoa have been exploited to great advantage in the understanding of molecular biology, the knowledge derived is conceptual to molecular biology and not restricted to protozoa. The reader is referred to molecular

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biology texts for information pertinent to that discipline and to standard biochemistry texts for corresponding information. Where the molecular biology is relevant to parasitic organism function as we understand it, it has been included and is, therefore, also distributed throughout the text where appropriate. Carbohydrate metabolism and energetics are given first consideration in the biochemistry discussions. More is known of these than other areas of biochemistry of these organisms. The aerobic and anaerobic metabolism of protozoa is contrasted with the fundamentally anaerobic metabolism of helminths. The theme of aerobic fermentation can be found among all of these organisms. This is followed by amino acid and protein metabolism; much new information on proteases is included in this section. In the chapter on purine and pyrimidine metabolism the common theme of de novo purine synthesis in protozoa is the framework upon which the species' variations are presented. Pyrimidine synthesis is more varied since effective salvage pathways exist in virtually all organisms. Polyamine metabolism, a basic discipline which, like purine metabolism, has emerged as a promising source of chemotherapeutic possibilities, is presented as a separate section. Lipid metabolism is understood best in relatively few species and, for this reason, we have elected to concentrate on these organisms rather than attempt to provide a compendium of information which cannot yet be understood in its biological context. Nucleic acid and protein synthesis are the only anabolic functions which have been kept separate. The others are captured within the foregoing chapters since they are integral to the catabolic activities of those pathways. Nucleic acid and protein synthesis provide compounds which are relatively stable and represent end-products in the organism. Antioxidant mechanisms and the metabolism of xenobiotics represent metabolism directed to the defense of the organism against exogenous chemicals. They are placed at the end of the sections of intermediary metabolism since they draw upon that knowledge. Antioxidant mechanisms have been given separate treatment because they have been well studied for a longer time, are more focused and, therefore, better understood. Cell surfaces are considered next in the transition from biochemistry into physiology. The membrane glycoproteins of protozoans are presented both with respect to their biochemistry and the dynamics of their insertion into this cellular structure. This is correlated with the section on lipid metabolism. Surfaces of helminths are next discussed in order to contrast these multicellular organisms with their single-celled counterparts. Cyst structures, although originally considered for inclusion, were reluctantly eliminated since the state of the art does not yet permit the correlations with biology and physiology that can be made for cell membranes. Cell organelles make an appearance as the volume progresses toward consideration of correlative multicellular physiology. These organelles provide many of the mechanisms which allow intracellular functions to occur and, thereby, permit intercellular activities to supervene. Some of the best understood aspects of physiology are in the neuromuscular systems of helminths and in their reproduction and development. Invasion mechanisms of protozoans are presented as an aspect of physiology of these cells since they require a co-opting of the biochemistry of the receiving organism and, therefore, an intercellular interaction. These conclude the integrative sections of the volume. The final chapter is a review of the foregoing information with respect to current chemotherapy and an attempt to predict where these basic sciences may be applied to medicine in the future.

PREFACE

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Immunological aspects of parasitology, a field which is both broad and detailed, has not been included. This was done in order to contain the size and maintain the focus of the volume. This has required that certain other important aspects of cell biology also be excluded, such as the antigenic shifts of trypanosomes. These are regrettable but necessary omissions and are well described in other texts of molecular biology and immunology. The biochemistry and cell biology of the variable surface glycoprotein have been included, however. We are proud of the group of collaborating authors in this volume. The knowledgeable scientist will recognize their contributions. We believe they have written clearly, comprehensively and well. Presentations by these seasoned investigators should be of interest to the experienced investigator, the graduate student and the newcomer. We must list first among the acknowledgements our authors. Each has provided contributions but also has reviewed the contributions of others. This has given an internal perspective which was of great assistance to the editors. External reviewers have been thoughtful and generous with their criticism and the volume has benefited accordingly. The patience of Academic Press and Dr Tessa Picknet in particular are gratefully acknowledged. Particular thanks are owed to Jean Smith, in Boulder, Colorado and to Karrie Polowetzky in New York who were of enormous assistance in organizing and preparing the manuscript. J. J O S E P H M A R R and

MIKLOS M ~ L L E R

Contributors

C. J. Bacchi Haskins Laboratories and Department of Biology, 41 Park Row, Pace University, New York, NY 10038-1502, USA R. L. Berens Division of Infectious Diseases, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA G. H. Coombs Parasitology Laboratory, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK R. E. Davis Department of Zoology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA R. Docampo Department of Veterinary Pathobiology, University of Illinois, Urbana, IL 61801, USA J. F. Dubremetz Unit6 42 INSERM, 369 rue J Guesde, 59650 Villeneuve d'Ascq Cedex-France R. H. Fetterer United States Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705, USA W. R. Fish Pediatric Endocrinology, State University of New York mHealth Science Center at Syracuse, 750 East Adams Street, Syracuse, NY 13210, USA H. R. Gamble United States Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705, USA T. G. Geary Animal Health Discovery Research, Upjohn Laboratories, Kalamazoo, MI 49001, USA B. G. Harris Department of Biochemistry and Molecular Biology, The University at North Texas, Health Science Center, 3516 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA R. Komuniecki Department of Biochemistry, University of Toledo, Toledo, OH 43605, USA E. C. Krug Division of Infectious Diseases, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA *B. T C. Lockwood Laboratory for Biochemical Parasitology, Department of Zoology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK *Died 8 October 1993.

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J. H. McKerrow Departments of Pathology, Medicine and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 and Department of Veterans Affairs Medical Center, San Francisco, CA 94121, USA J. J. Mart Ribozyme Pharmaceuticals Inc., 2950 Wilderness Place, Boulder, CO 80301, USA M. Miiller The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA T. Nilsen Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA M. J. North Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, UK F. R. Opperdoes International Institute of Cellular and Molecular Pathology and University of Louvain, ICP-TROP/7439, avenue Hippocrate. 74, B-1200 Brussels, Belgium M. Parsons Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA 98109, and Department of Pathobiology, University of Washington, Seattle, WA 98195, USA A. O. W. Stretton Department of Zoology, University of Wisconsin-Madison, Madison, WI 53706, USA D. P. Thompson Animal Health Discovery Research, Upjohn Laboratories, Kalamazoo, MI 49001, USA J. W. Tracy Departments of Comparative Biosciences and Pharmacology, and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WI 53706-1102, USA S. J. Turco Department of Biochemistry, University of Kentucky Medical Center, Lexington, KT 40536, USA B. Uliman Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA E. Uilu Department of Internal Medicine and Cell Biology, Yale University School of Medicine, 333 Cedar Street/LCI 801, New Haven, CT 06520-8022, USA J. F. Urban United States Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Parasite Immunobiology Laboratory, Beltsville, MD 20705, USA E. A. Vande Waa Department of Comparative Biosciences, University of WisconsinMadison, 2015 Linden Drive West, Madison, WI 53706-1102, USA N. Yarlett Haskins Laboratories and Department of Biology, 41 Park Row, Pace University, New York, NY 10038-1502, USA

Molecular Biology of Protozoan and Helminth Parasites ELISABETTA ULLU ~ and T I M O T H Y NILSEN 2

~Department of Internal Medicine and Cell Biology, Yale University School of Medicine, New Haven, C T and 2Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, USA

SUMMARY

The intent of this chapter is to offer an overview of selected aspects of the molecular biology of parasitic protozoans and helminths. Important topics, i.e. DNA rearrangements associated with antigenic variation in protozoans and chromosome diminution in nematodes have been omitted. Nevertheless, it should be apparent that molecular analysis of parastic organisms has been remarkably productive in revealing unusual and unexpected pathways of gene expression. We emphasize that, although such phenomena as trans-splicing and RNA editing were discovered in parasites, they are not restricted to parasites, and thus cannot be considered adaptations to parasitism. This in no way diminishes the importance of the discoveries and there is every reason to suspect that further investigations in parasitic organisms will continue to provide novel insights into mechanisms of eukaryotic gene expression in general while simultaneously suggesting targets for chemotherapeutic intervention.

1. INTRODUCTION

Within the last ten years, the application of modern molecular biological approaches has provided a wealth of knowledge regarding gene structure, organization and expression in parasitic organisms. It is difficult to provide broad coverage of all topics Biochemistry and Molecular Biology of Parasites ISBN 0-12-473345-X

Copyright 9 1995 Academic Press Ltd All riohts of reproduction in any form reserved

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of interest in protozoan and helminth molecular biology. This discussion is restricted to trypanosomatids as 'representative' protozoans and nematodes as 'representative' helminths. This choice is dictated by the fact that these organisms, because of their experimental tractability, have been most fruitful in yielding biochemical insight into parasite gene expression. The three most important areas, transcription, trans-splicing and RNA editing were selected because, in addition to providing important insight into parasite gene expression, they directly impinge upon our understanding of the molecular biology of eukaryotic cells in general. In this regard, basic principles of molecular biology will not be discussed. The reader is referred to any of several excellent textbooks for appropriate background.

2. TRANSCRIPTION IN TRYPANOSOMES AND NEMATODES 2.1. Trypanosome Gene Expression (an Overview) Compared to other eukaryotic organisms, trypanosomes are unusual in that they contain a number of tandem arrays of genes coding for housekeeping and developmentally regulated proteins, i.e. tubulins, calmodulin, ubiquitin, surface antigens, glycolytic enzymes. Although most protein coding genes appear to be repetitive, some single copy genes have been identified. The spacing between protein coding regions is quite short and ranges from less that one hundred to a few hundred nucleotides. This appears to be the case between repeating units within a gene cluster and also between genes encoding different proteins. This degree of packaging of genetic information is somewhat higher than in other unicellular eukaryotic organisms of similar genetic complexity like the yeast Saccharomyces cerevisiae.

FIG. 1.1 Biogenesisof trypanosomatid mRNA. A schematic view of trypanosome gene expression. Mature mRNAs are generated from polycistronic pre-mRNAs via two RNA processing reactions, trans-splicingand polyadenylation. For details, see text.

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Messenger RNA molecules of trypanosomatids are chimaeras of two different gene transcripts, the SL (spliced leader) RNA, which contributes the 5' end of the mature mRNA, and the pre-mRNA, which provides the body of the messenger RNA coding for the various proteins; joining of these molecules occurs by trans-splicing (Fig. 1.1). Thus, in trypanosomatids, the 5' end of mRNA is formed by an RNA processing reaction rather than by transcription initiation as is the case in most higher eukaryotes. In this context trans-splicing can be viewed as an alternate and unusual pathway of mRNA 5' end formation. The formation of trypanosome mRNA 3' ends is thought to occur, as it does in higher eukaryotes, through an RNA processing reaction which involves cleavage at the polyadenylation site with the concomitant addition of a poly(A) tail to the newly formed mRNA 3' end. However, no consensus sequence similar to the AAUAAA polyadenylation signal has been identified in trypanosomatid mRNAs and it is at present unclear whether specific pre-mRNA signals are required for mRNA 3' end formation. In summary, the biogenesis of mature trypanosome mRNA takes place exclusively via RNA processing and requires two cleavage reactions of the pre-mRNA, one at the splice acceptor site (discussed in section 3 below) and the other at the site of addition of the poly(A) tail.

2.2. Trypanosomatid Genes Are Organized as Polycistronic Transcription Units Soon after it was realized that the 5' end of mature mRNA is produced by RNA processing, the structure of the mRNA primary transcripts which are the substrates for trans-splicing became a major point of interest. In particular, because of the tandem reiteration of many genes and the small size of intergenic sequences, it was suggested that trypanosomatid transcription units might encompass more than one gene and that the corresponding primary RNA transcripts might be polycistronic. Indeed a considerable amount of evidence accumulated suggesting that this was the case (1-3). In particular: (i) nuclear run-on assays and nuclease protection assays of newly synthesized RNA in vivo showed that intergenic regions are continuously transcribed together with the protein coding regions; (ii) the kinetics of UV inactivation were consistent with the presence of a promoter sequence located upstream of large chromosomal regions which included several genes; (iii) dicistronic and polycistronic transcripts were identified in steady-state RNA and their accumulation was increased by treatments which inhibited trans-splicing. However, all the above evidence was rather indirect. Convincing proof that polycistronic transcription units could be functional came when efficient DNA transfection and expression systems were established for a number of trypanosomatids. Using synthetic constructs under controlled conditions, it has been demonstrated that intergenic regions from a number of gene clusters do not possess detectable promoter activity. However, the same intergenic regions, when sandwiched between two reporter genes in the form of dicistronic transcription units under the control of the PARP promoter (see below), provide RNA processing signals of polyadenylation and transsplicing of the upstream and downstream mRNA, respectively. Although dicistronic and polycistronic mRNAs can be identified in steady-state RNA, two lines of evidence suggest that these molecules probably are not authentic precursors to mature mRNAs. First, in Trypanosoma brucei cells, trans-splicing at three different splice acceptor sites (the ~ and fl tubulin, and the actin RNA coding regions),

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occurs within less than one minute from the time the polymerase has traversed the splice site (4). Considering the average rate of elongation of RNA polymerase II, it is almost certain that trans-splicing of pre-mRNA is accomplished co-transcriptionally. Second, in isolated tryanosome nuclei it appears that cleavage at the polyadenylation site of a heat shock protein (HSP 70) pre-mRNA occurs on nascent RNA chains (5). Thus, it is unlikely that polycistronic pre-mRNAs are synthesized in toto. In light of these findings the dicistronic and polycistronic mRNAs detected in steady-state conditions most likely represent aberrant products which have failed to undergo complete processing by the trans-splicing and polyadenylation machineries. Polycistronic transcription units are typical of prokaryotic organisms and of some eukaryotic viruses. Trypanosomes (and nematodes which also trans-splice, see below) represent the only examples of eukaryotes which transcribe their genes in this way. At present, there is no evidence that the various genes encoded in polycistronic transcription units in trypanosomes are related in their function as are operons in bacteria. Likewise, we do not know what the average size of a transcription unit is in trypanosomatids and whether there is any attenuation of transcription in regions that are promoter-distal relative to those that are promoter-proximal. This latter consideration has obvious relevance for the control of gene expression in these organisms.

2.3. Promoter Structure of Trypanosome Protein Coding Genes Transcription of most protein coding genes in eukaryotic cells, including trypanosomes, is carried out by RNA polymerase II in concert with a variety of general and specific transcription factors. However, in African trypanosomes there are two known exceptions to this rule: the gene families coding for the variant surface glycoproteins (VSG) of bloodstream forms and the Procyclic Acidic Repetitive Protein (PARP). Whereas RNA polymerase II-mediated transcription is sensitive to low concentrations of the elongation inhibitor ~-amanitin, transcription of both VSG and PARP genes is resistant to high concentrations of the inhibitor. This initial finding, corroborated by other lines of evidence, supports the notion that RNA polymerase I, the same RNA polymerase that transcribes ribosomal RNA genes, is responsible for transcription of these two gene families (6). However, the lack of an in vitro transcription system precludes at present the unequivocal identification of RNA polymerase I as the relevant polymerase of VSG and PARP gene transcription. Notwithstanding this ambiguity, the early analysis of the mode of transcription of these two gene families combined with recent advances at introducing foreign DNA into trypanosomatids, have provided the basis for the conclusive identification of promoters in T. brucei. Cis-acting DNA sequences which are necessary and sufficient for transcription initation of these gene families (promoters) have been identified. For the VSG locus the promoter sequences lie approximately 4 5 - 4 0 k b upstream from the VSG gene (7) whereas the PAPP promoter is within a few hundred nucleotides upstream of its mRNA body. A detailed mutational analysis of the PARP promoter sequence has revealed that the promoter structure is complex, composed of at least three sequence elements located within 250bp 5' to the transcription start site (8, 9). The topological arrangement of both of these sequence elements (whose integrity is essential for promoter activity) in relation to each other and to the transcription start site is reminiscent of typical promoters for RNA polymerase I.

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Mutational analysis of the PARP promoter showed that promoter sequences and signals required for trans-splicing of the pre-mRNA do not overlap. These observations stimulated a series of elegant experiments which demonstrated that a chimaeric transcription unit consisting of the T. brucei ribosomal RNA promoter linked to a 3' splice site acceptor region and followed by a reporter gene is efficiently transcribed and trans-spliced in T. brucei cells (6). These are the only known examples of RNA pol I-mediated expression of eukaryotic protein coding genes and they underscore the possibility that some endogenous trypanosome genes, like the VSG and the PARP genes, might indeed be transcribed by RNA polymerase I. Although in the past years, much has been learned about the structure of VSG and PARP gene promoters, at present essentially nothing is known about the promoter structure of the majority of protein coding genes in trypanosomatids. These are the genes which are transcribed by an RNA polymerase with the a-amanitin sensitivity typical of other eukaryotic RNA polymerase IIs. Thus far, experiments aimed at measuring the density of RNA polymerase molecules along chromosomal regions transcribed by RNA polymerase II have failed to detect discontinuities in the transcription maps which would be consistent with the presence of promoter sequences. Furthermore, attempts to demonstrate promoter activity in trypanosome DNA sequences located immediately upstream or in between genes of a tandem array have met with no success. The available evidence, although of a negative nature, points to the possibility that promoters for RNA polymerase II might be quite sparse in the trypanosomatid genome. A more provocative, but perhaps less likely, interpretation is that typical RNA polymerase II promoter sequences could be absent from trypanosomatid DNA. However, if this were true, how would RNA polymerase II initiate transcription? Possibly, the transcription complex has little, if any, sequence specificity and could initiate transcription at many different locations along chromosomal DNA. Such an explanation could account for the homogeneous rates of transcription observed at several chromosomal loci.

2.4. Regulation of Gene Expression in Trypanosomes Although it is not known for sure how many RNA polymerase II promoters exist in the genome of trypanosomatids, it is now generally accepted that the general mode of transcription of both housekeeping and developmentally regulated protein coding genes is polycistronic. Since for most protein coding genes there is no evidence of transcriptional control, how is gene expression regulated in trypanosomatids? Relevant to this is the observation that in steady-state conditions the cellular representation of different mature mRNAs (even those derived from the same transcription unit) can vary widely. The best studied example comes from the VSG transcription unit, which consists of the VSG gene and several upstream genes. Here mRNAs encoded from the upstream genes are much less abundant than the VSG mRNA itself. Since nuclear run-on experiments indicate that the rate of transcription along the VSG transcription unit is quite homogeneous, it has been inferred that mRNA abundance is primarily regulated by post-transcriptional mechanisms (10). These could include pre-mRNA stability, transsplicing or mRNA 3' end maturation (see Fig. 1.1). Each of these steps could conceivably be regulated and could modulate the output of mature mRNA from a given

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gene. Indeed, differential mRNA stability has been proposed to be a major factor in the developmental regulation of the VSG and PARP gene products during differentiation of African trypanosomes. At present there is no direct evidence that trans-splicing is a regulating process nor that the rate of trans-splicing varies between different splice acceptor sites. The same is true for the process of mRNA 3' end formation. However, some experiments support the notion that these post-transcriptional mechanisms may play a pivotal role in regulating gene expression in trypanosomatids. For instance, it has been demonstrated that when trans-splicing is inhibited by destroying either U2, U4 or U6 snRNAs (see below), unspliced tubulin pre-mRNA is rapidly degraded, possibly because the 5' end of newly synthesized pre-mRNA is not capped (4). These findings suggest that the output of mature mRNA from the corresponding pre-mRNA could be determined by the balance between the rate of pre-mRNA degradation and the rate of trans-splicing at a given splice acceptor site in the pre-mRNA. Further support for this notion comes from the observation that inhibition of trans-splicing results in almost complete inhibition of the formation of the 3' end of mature mRNA molecules (11). This finding has led to the hypothesis that in trypansomatids there might be a hierarchical order in the pathway of pre-mRNA processing reactions. In this model trans-splicing, or perhaps assembly of a trans-splicing complex on the premRNA, is a prerequisite for subsequent recognition of the mRNA 3' end formation and polyadenylation signals. An alternative and intriguing possibility is that in the premRNA the signals required for trans-splicing partially or perhaps completely overlap with those determining the choice of the polyadenylation sites.

2.5. Transcription of Nematode Protein Coding Genes The discussion above should make it clear that there are significant gaps in our understanding of transcription in trypanosomatids. Unfortunately, even less is known regarding transcriptional control elements and transcription units encoding mRNAs in nematodes. Only a few promoters have been tentatively mapped and this work was carried out exclusively in the free-living nematode, Caenorhabditis elegans. Our knowledge of promoters and transcription units in other nematodes is non-existent. However, there are intriguing parallels between trypanosome gene expression and nematode gene expression which suggest that lessons learned in trypanosomes may be applicable to nematodes. Nematodes also carry out trans-splicing and recent evidence from work in C. elegans suggests that trans-splicing may serve the same function as it does in trypanosomes [i.e. processing polycistronic transcription units (12)]. In C. elegans two distinct SL RNAs are used for trans-splicing (SL1 and 2). Investigations designed to determine the specificity of SL1 or SL2 addition revealed that SL2-accepting pre-mRNAs were encoded by genes that were located only a short distance (-~ 100nt) 3' of upstream genes transcribed in the same orientation. Transgenic analysis using a specific gene pair demonstrated that promoter elements driving expression of the SL2 accepting gene were not present in either the intergenic region or in the upstream gene itself. Instead expression of both genes was driven by elements well 5' of the upstream gene. In additional experiments, a heat shock promoter was inserted immediately in front of the gene pair. In this case, mature message for the downstream gene was made, it contained the SL2 leader sequence, and its appearance

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was dependent on heat shock. This experiment demonstrated that when a polycistronic RNA was created artificially, it was capable of yielding mature, correctly trans-spliced mRNA. From these and other analyses, it seems clear that polycistronic transcription units exist in C. elegans. Furthermore, it appears that a major (if not the only) determinant of SL2 addition in C. elegans is that the accepting pre-mRNAs be encoded by genes internal in such transcription units. These studies may have a broader significance to our understanding of the biological role of nematode trans-splicing in general. In this regard, it seems that the demonstration of polycistronic transcription units in nematodes is of fundamental importance. If such transcription units are common in nematodes, it suggests that the major function of trans-splicing in these organisms (as in trypanosomes) is in the maturation of 5' ends of mRNAs located within long poly-pre-mRNAs. In this view, SL2 trans-splicing in C. elegans would reflect a specialized form of trans-splicing which is used only when adjacent genes are in close proximity to each other. SL1 trans-splicing in C. elegans, and trans-splicing in other nematodes [which apparently lack alternative SL RNAs (see section 3)] would be used when adjacent pre-mRNAs are more widely spaced in the primary transcript. Although this notion has intrinsic appeal in that it would provide a functional link between trans-splicing in nematodes and trypanosomes, it remains largely speculative. Clearly, systematic transcription unit mapping is necessary before any conclusions can be drawn regarding the role (if any) of nematode trans-splicing in processing polycistronic mRNAs.

2.6. Transcription of the Nematode SL RNA In contrast to our dearth of knowledge regarding promoters for mRNAs in nematodes, we have a detailed understanding of transcription of nematode trans-spliced leader RNAs. These RNAs resemble the U snRNAs (see section 3) which are required for cis-splicing and are transcribed by RNA polymerase II. Dissection of the SL RNA gene promoter became possible when it was shown that the Ascaris SL RNA gene was accurately and efficiently transcribed by RNA polymerase II from cloned templates in Ascaris embryo extracts (13, 14). This cell-free system permitted the identification of SL RNA transcriptional control elements through mutational analysis. These experiments revealed that the SL RNA gene represents an unusual RNA polymerase II transcription unit. Efficient initation of SL RNA synthesis required two sequence elements, one of which was centered approximately 50 nucleotides upstream from the transcriptional start site. Remarkably, the second sequence element was the 22nt SL sequence itself; mutations in the SL sequence abolished transcription in vitro. DNase I footprinting showed that the SL sequence bound a protein factor; the boundaries of the footprint exactly coincided with the SL sequence. Competition experiments indicated that binding of the factor was directly correlated with transcription (13). Further analysis has indicated that the 22nt binding factor is ~ 60 kDa protein. It will be of considerable interest to see if this factor shares any homology with known transcription factors involved in RNA polymerase II transcription. Additional experiments characterizing SL RNA transcription showed that 3' end formation (termination) of SL RNA synthesis in vitro was also unusual in that it

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depended exclusively upon gene-internal sequences. This contrasts to the situation in vertebrate U snRNA genes where the primary determinant of 3' end formation is the so called 3' end formation box located a short distance downstream of mature 3' ends (15,16). The organization and expression of the SL RNA gene has some interesting implications. The nematode SL sequence has been perfectly conserved in widely diverged nematodes. This conservation was commonly interpreted to mean that the 22nt SL sequence present on trans-spliced mRNAs must have some important posttranscriptional function. The finding that the SL sequence is an essential promoter element for its own synthesis suggests an alternative explanation for sequence conservation, i.e. the conservation could be dictated by constraints imposed by the binding specificity of a transcription factor.

3. TRANS-SPLICING 3.1. Overview Intermolecular (trans)-splicing is, by definition, an RNA processing reaction which precisely joins exons derived from separately transcribed RNAs. Two types of transsplicing are known to occur in nature. The first type is directly analogous to the cis-splicing of group II introns and is confined to 'split' introns in organelles of plants and fungi. The second type is analogous to the snRNP-mediated removal of introns from nuclear pre-mRNAs and is known as spliced-leader addition trans-splicing (17-20). This type of trans-splicing has been demonstrated in a variety of lower eukaryotes including trypanosomatids, Euolena, trematodes and nematodes (21-23). In the trypanosomes all pre-mRNAs acquire their 5' terminal exon (the spliced leader, SL) from a small RNA termed the SL RNA. The evidence suggests that there are no conventional (cis) introns in trypanosomes (17). In the other organisms that carry out spliced leader addition trans-splicing, only a subset of pre-mRNAs receive the SL. In further contrast to trypanosomes, trans-spliced pre-mRNAs in these organisms contain conventional introns which are processed by cis-splicing. The removal of intervening sequences from pre-mRNAs proceeds through two consecutive transesterification reactions. In the first step, the 3'-5' phosphodiester bond at the splice donor site is cleaved, releasing the 5' exon. Concomitant with this cleavage is the formation of a 2'-5' phosphodiester bond between the 5' end of the intron and an adenosine residue about 30nt upstream of the splice acceptor site. The resultant intermediate is known as the lariat or 2/3 intermediate. The second step of splicing also involves the cleavage and concomitant ligation of phosphodiester linkages. The 3' end of the 5' exon generated in step 1 attacks the phosphodiester bond at the splice acceptor site resulting in two ligated exons and the release of the intron in the form of a lariat. Subsequent to intron release, the 2'-5' bond is cleaved by an enzyme known as a debranching enzyme and the resultant linear intron is degraded in the nucleus. Trans-splicing follows the same two steps (Fig. 1.2). In the first step the SL (5' exon) is cleaved from the rest of the SL RNA. Concomitant with this cleavage is formation of a 2'-5' phosphodiester bond between the 'intron' portion of the SL RNA and the

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FIG. 1.2 Cis and trans-splicing proceed through analogous two-step reaction pathways. A schematic illustration of the similarities between cis and trans-splicing. See text for details. branch site. Because the SL RNA the intermediate generated by the instead of a lariat (24, 25). In the attacks the acceptor site, yielding which is subsequently debranched

and pre-mRNA are encoded as separate molecules, first step of trans-splicing is a Y branched structure second step of trans-splicing the 3' OH on the SL ligated exons and releasing the Y branch structure and degraded.

3.2. Mechanism of Trans-Splicing: the Spliced-Leader RNAs Uridine-rich Small Nuclear RNAs (U snRNAs) are abundant short RNAs in eukaryotic cells. A subset of these RNAs participates in the catalysis of splicing (see below). The spliced leader RNAs (SL-RNAs) of both trypanosomes and nematodes bear striking similarities to the U snRNAs required for cis-splicing (Fig. 1.3.) This is particularly clear in nematodes where the SL RNA possesses both a trimethylguanosine cap structure and an Sm binding site (18,20). The Sm binding site of snRNAs conforms to the consensus sequence RR UnRR (where R is a purine) and is located in a single-stranded region between two stem loops. In U snRNAs the Sm binding site is necessary for assembly of snRNAs into snRNPs (small nuclear ribonucleoproteins) because it promotes the binding of core Sm proteins (26, 27). Several years ago a number of groups demonstrated that the Sm binding sequence of the C. elegans SL RNA was functional since the C. elegans SL RNP was precipitable from C. elegans extract with human Sm antisera. Furthermore, the SL RNA became precipitable with Sm antisera when incubated in HeLa cell extracts (28). It was subsequently established that assembly of the Ascaris SL RNA into an Sm snRNP was an absolute prerequisite for its participation in trans-splicing in vitro (see below) (29). The relationship between the trypanosome SL RNA and the trypanosome U snRNAs was less obvious, since proteins associated with U snRNPs in trypanosomes do not contain epitopes recognized by Sm antisera. Furthermore, the trypanosome SL

10

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A N D T. N I L S E N

FIG. 1.3 SL RNAs have similar structures and resemble U snRNAs. Schematic representation of secondary structures of SL RNAs from organisms known to carry out trans-splicing. See text for details.

RNA does not have the T M G cap characteristic of U snRNAs but instead has a monomethylated guanosine as the capping nucleotide. This RNA also has a highly unusual array of base modifications in its first few nucleotides, the structural details of which have only recently emerged (30). However, the relationship between the trypanosome SL RNP and U snRNPs has been clarified recently with the successful affinity

1. MOLECULAR BIOLOGY OF PROTOZOAN AND HELMINTH PARASITES 11 purification of SL RNPs and snRNPs from T. brucei (31). These analyses have shown convincingly that the SL RNA and snRNAs in trypanosomes share a common set of core proteins resembling the core proteins of other eukaryotic Sm snRNPs. Collectively, these observations have reinforced the hypothesis originally proposed (32) that SL RNAs represent a special type of U snRNP where an exon (the SL) is fused to an snRNA-like sequence. Unlike the snRNAs required for cis-splicing, the SL RNA is consumed during trans-splicing. An intriguing question that remains the topic of speculation is whether SL RNAs predated the snRNAs as we know them today or alternatively arose from the union of a pre-existing snRNA and a free exon (33).

3.3. Features of the Nematode SL RNA Essential for Function in Trans-Splicing An homologous cell free system which utilizes synthetic SL RNA (generated by in vitro transcription) in trans-splicing has permitted a detailed dissection of the nematode SL RNA (34). Site-directed mutagenesis and chemical modification interference studies have revealed that critical functional elements in the SL RNA are confined to remarkably short regions of the molecule, all of which reside in the snRNA-like domain (35). Surprisingly, exon sequences (the 22 nt SL) are irrelevant for SL RNA function in trans-splicing in vitro (36). The fact that the 22 nt SL was not important for SL RNA participation in trans-splicing was not anticipated since this sequence has been stringently conserved in evolutionarily distant nematodes. However, this sequence conservation can be explained in part by the fact that at the DNA level, the SL sequence itself is a promoter element essential for SL RNA synthesis by RNA polymerase II (see above). Within the snRNA-like domain, essential sequences are confined to the single stranded region between the second and third stem loops. This sequence contains the Sm binding site and adjacent nucleotides. Mutational analyses have shown that there is an absolute correlation between the capacity of the SL RNA to assemble into an Sm snRNP and its ability to function in trans-splicing. Thus, it is clear that the SL RNA participates in trans-splicing as an Sm snRNP. The Sm binding site is critical for SL RNA function; cap structure, which constitutes the other similarity between SL RNAs and U snRNAs, is not. Although the trimethylguanosine cap structure is clearly not required for SL RNA function in trans-splicing in vitro, it may be required for proper subcellular localization of the SL RNP in vivo. Studies in other systems have demonstrated that the TMG cap on U snRNAs is part of bipartite nuclear localization signal (27). The lack of SL RNA cap recognition in nematode trans-splicing stands in apparent contrast to recent results obtained in trypanosomes. Two groups have shown that trypanosome SL RNAs, undermethylated in the cap structure, do no function in trans-splicing. These results suggest that the unique trypanosome SL RNA cap may be required at some point in the assembly of the trans-spliceosome. However, this interpretation is not clear, since it is possible that, as with other snRNAs, the cap could serve to localize the trypanosome SL RNA to the nucleus. Until the pathway of SL RNP assembly is worked out or a trypanosome cell free system is established this will remain an open question. Nevertheless, the enzymes that catalyze cap modification in

12

E. ULLU AND T. NILSEN

trypanosomes present themselves as attractive candidates for chemotherapeutic intervention since such modification is clearly required for productive use of the trypanosomatid SL RNA.

3.4. Significance of Essential Sequences Within the Nematode SL RNA Functionally relevant sequence elements within the nematode SL RNA are confined to the splice donor site and the Sm binding region. Although it is clear that the Sm binding site is required for assembly of the SL RNA into an Sm snRNP, recent experiments have shown that this single-stranded region may have an additional role in transsplicing. Functional Sm binding sequences, derived from other U snRNAs, fail to support SL RNA function when substituted into the SL RNA: this indicates that the primary sequence of the Sm binding region of the SL RNA is important. Such sequence constraints can be imposed for a variety of reasons, one of which is the necessity to interact by base-pairing with another RNA. Indeed, crosslinking experiments have shown that the SL RNA interacts with U6 snRNA via a base-pairing interaction. In the SL RNA the base-paired region spans the sequences critical for its function (35). In U6 snRNA, the region of base pairing adjoins and overlaps the region of U6 known to interact with U2 snRNA in higher eukaryotes and in trypanosomes (37,38). While the functional significance of the SL/U6 interaction remains to be established experimentally, it may be that this R N A - R N A interaction facilitates entry of the SL RNA into the trans-spliceosome. If true, it would provide some insight into one of the fundamental questions in trans-splicing; i.e., how do the two substrates (the SL RNA and pre-mRNA) efficiently associate within the nucleus in the absence of significant sequence complementarity?

3.5. Mechanism of

Trans-Splicing: Required snRNP Cofactors

Conventional cis-splicing requires the participation of five U snRNPs, U I, U2, U4, U6 and U5 (39). U1 snRNP is involved in 5' splice site identification and recent evidence suggests a role for U1 snRNP in acceptor site recognition as well. U5 snRNP has also recently been shown to be involved in splice site recognition whereas U2 snRNP identifies the branch point on the pre-mRNA. U4 and U6 join the spliceosome together as a base-paired double snRNP which does not appear to recognize specific features of the pre-mRNA. U6 may be a catalytic RNA which promotes the actual splicing of exons. See reference (40) for recent discussion and illustration of R N A - R N A interactions in cis-splicing. Trypanosomes contain homologs of U2, U4 and U6 snRNAs (41, 42). RNase H degradation studies showed that these snRNAs are required for trans-splicing (4). To date no homologs of U1 or U5 snRNAs have been found in trypanosomes, leading to the notion that neither of these snRNAs is required for trans-splicing. If true (and evidence against a role for U 1 and U5 in trans-splicing in trypanosomes is exclusively negative) it would suggest that trans-splicing is significantly simpler than cis-splicing, but would leave the problem of how the 5' and 3' splice sites are juxtaposed. A detailed discussion of the role of U snRNAs in trans-splicing is contained in ref (20). Since nematodes carry out both cis- and trans-splicing, it is not surprising that they contain a complete complement of U snRNAs including U1 and US. In vitro analysis,

1. MOLECULAR BIOLOGY OF PROTOZOAN AND HELMINTH PARASITES 13 using targeted degradation has shown that, as in trypanosomes, U2, U4 and U6 are required for trans-splicing. To date, it has not been possible to unambiguously determine the role or lack thereof of U1 or U5 snRNAs in nematode trans-splicing. This is clearly an important question since the answer will provide significant insight into the similarities or differences between trans-splicing in nematodes and trypanosomes.

3.6. Biological Roles of 5' Leader Addition by Trans-Splicing As discussed above, trans-splicing serves to mature 5' ends of mRNAs embedded with polycistronic transcription units in trypanosomes and may serve the same function in nematodes. However, trans-splicing may be important in other aspects of mRNA metabolism. In addition to providing a discrete 5' end for mRNAs, trans-splicing also provides each mRNA with the cap structure derived from the SL RNA. Although it remains to be established whether translation in trypanosomes or nematodes is cap dependent, it seems likely that the cap, as it is in other eukaryotes, will be essential for mRNA recognition by ribosomes. It remains to be determined whether spliced leader or cap addition is necessary for transport of mRNAs from the nucleus or influences mRNA stability. Because not all nematode mRNAs acquire the SL (43), trans-splicing might be a regulatory mechanism in these organisms. However, work in several laboratories has failed to reveal that trans-splicing of any pre-mRNA is regulated in a developmental, tissue-specific or stess-induced fashion. Furthermore, there is no suggestion that trans-splicing is restricted to a group of mRNAs which encode functionally related proteins. Much of the impetus for seeking a regulatory function for trans-splicing arose from the perception that only a minority (10-15%) of nematode messages was trans-spliced. However, this may have been an underestimate. The percentage of trans-spliced mRNAs was initially arrived at by interpretation of hybrid-arrest translation experiments carried out in rabbit reticulocyte lysates programmed with C. elegans mRNAs. Subsequent experiments demonstrated that trans-spliced nematode messages retained the SL RNA's trimethylguanosine cap structure (see above). Since it has been established that reticulocyte lysates translate trimethylguanosine-capped messages very poorly it seems probable that, in the early experiments proteins encoded by transspliced mRNAs could have been underrepresented in total translation products. Hybrid-arrest translation experiments indicate that 80-90% of Ascaris mRNAs are trans-spliced. If these findings can be generalized to other nematode species, they would indicate that trans-splicing plays a much more central role in nematode gene expression than was previously suspected.

4. RNA EDITING 4.1. Editing, the Phenomenon In trypanosomatids the mitochondrial DNA [kinetoplast DNA (kDNA)] consists of two sets of circular molecules, namely 20-50 maxicircles (20-39kb in size) and 5000-10000 interlocked minicircles (0.5-2.5 kb in size). The maxicircles are analogous

14

E. ULLU AND T. NILSEN EDITED

COIII

mRNA:

s'... AuCCAGuAuuGuu~u~uAuGG~u~u~A~GuAG|

]...3'

l-IJJ.'-lJIIJJ--lll. Jli..JII.IlllJJillllfl gRNA

Tbl:

3 ' . . . nU u U A U U A U A G U G A A A A A U G U C A A G A A U G U A U C G C U C A A A C A A A A U A U A U A . . .

lJl 5 '

Anchor

FIG. 1.4 An example of kinetoplastid pan-editing. A portion of the mature T. brucei cytochrome oxidase subunit III is shown above a guide RNA. Uridines inserted via editing are shown in lower case. Insertion of the uridines within the box is directed by a different guide RNA not shown. For details see text and ref. (45).

to the mitochondrial DNA of other eukaryotes and code for mitochondrial rRNAs and for some of the protein components of the respiratory chain. Sequencing of maxicircle DNA from various species brought about the remarkable discovery of kinetoplast RNA editing, one of the most astonishing findings in the field of RNA processing (44). Initial analysis revealed that the gene coding for cytochrome oxidase subunit II was interrupted by a frameshift; however, the frameshift was eliminated by insertion of four uridine residues in the corresponding mRNA. Since this early report, a wealth of information has been accumulated on the phenomenon of kinetoplast RNA editing which involves the non-encoded addition and, to a lesser extent, deletion of uridine residues in kinetoplast mRNAs. RNA editing not only eliminates internal frameshifts but also provides otherwise untranslatable open reading frames with the initiator codon AUG. Most startling was the discovery that editing can be so extensive as to remodel more that 50% of the mRNA sequence (pan-editing). Examples of the pan-editing occur in the cytochrome oxidase subunit III, N A D H dehydrogenase and ATPase 6 mRNAs of T. brucei (Fig. 1.4). The process of editing is post-transcriptional and seems to occur in a 3' to 5' direction. Directionality of editing is suggested by the sequence analysis of many partially edited mRNAs (perhaps editing intermediates) which contain edited 3' sequences and unedited 5' sequences. Editing is quite specific since it occurs only in certain regions of a transcript and is also remarkably precise since translatable open reading frames are generated.

4.2. Mechanism of RNA Editing The existence of editing raises important fundamental questions: for example, where is the information for the edited sequences stored and how is the information transferred to the mRNA. A possible answer to the first question came from the discovery of small kinetoplast RNAs ( 6 0 - 7 0 n t in size), termed guide RNAs (gRNAs) (Fig. 1.4), that are encoded in both minicircle and maxicircle DNA. The main features of gRNAs are as follows, gRNAs contain at their 5' end a short anchor sequence complementary to a region immediately 3' of an edited site. This is followed by a sequence which is complementary to the edited version of the mRNA sequence. At the 3' end of the gRNAs there is a polyU tail which is added post-transcriptionally. Thus, gRNAs appear well suited to store the information for the edited sequence and must be important in the editing pathway. How the information contained within the guides is transferred from gRNAs to mRNAs is still an open question. Two models have been proposed to

1. MOLECULAR BIOLOGY OF PROTOZOAN AND HELMINTH PARASITES 15 account for the editing pathway, the so called enzyme cascade and the transesterification models. At present, experimental evidence to distinguish between these hypotheses is lacking. To account for the structure of the edited sequences, primary RNA transcripts (pre-edited RNAs) must undergo a number of cleavage and ligation reactions at the editing sites with concomitant addition or deletion of U residues. The transesterification model is at present favored because of its intrinsic simplicity and its analogy to the chemistry of RNA splicing. It is proposed that editing is achieved through multiple rounds of two-step transesterification reactions. In the first step the gRNA base pairs to the pre-edited site positioning the 3' OH group of the terminal uridine for attack on the phosphodiester bond at the editing site. Such a reaction would produce two intermediates, a free 5' fragment of the pre-edited RNA cleaved at the edited site and a chimaeric molecule consisting of the gRNA covalently linked to the 3' portion of the pre-edited RNA. In the second step, the 3' OH of the pre-mRNA 5' fragment would attack the chimaeric molecule, with concomitant ligation of the 5' and 3' end fragments of the pre-mRNA. Depending on the precise location of this second nucleophilic attack, the pre-mRNA would have one or more Us inserted or deleted at the editing site and the gRNA would be shortened or lengthened accordingly. This process would presumably be repeated until the edited site was fully base-paired to the complementary sequences in the gRNA. Experimental support for this model comes from the finding that chimera-forming activities are indeed present in kinetoplast extracts and that chimeric molecules can be detected in vivo. However, this evidence can not be considered definitive since chimera formation could conceivably be accomplished by a battery of enzymatic activities. Such enzymes would include an endonuclease specific for the edited sites, an RNA ligase and a terminal uridylyl-transferase. Each of these activities has been demonstrated in kinetoplast extracts. Whether or not enzyme activities are required for the editing pathway will become clear once an in vitro system capable of carrying out a complete editing cycle is available. It should be stressed that all the available evidence about putative intermediates in the editing pathway has been obtained from inspection of cDNA molecules copied from cellular RNAs in steady-state condition. Although it is commonly assumed that these molecules are bona fide intermediates in the editing pathway, the possibility exists that at least some of them could be aberrant processing products. From all these studies, it has become apparent that the process of editing is incredibly complex. This is especially true for the cases of pan-editing, like that observed in the cytochrome oxidase subunit III mRNA of T. brucei. Many different gRNAs are required for this process and they must be utilized sequentially presumably in a 3' to 5' direction. How these different gRNA molecules are specifically recruited and in which order they act is the subject of intense investigation. In addition to mechanistic questions, the existence of RNA editing adds an unanticipated layer of complexity to our understanding of gene expression. Editing clearly violates the 'central dogma' of molecular biology and examples of RNA editing have now been found in diverse species from humans to slime molds. Time will tell how widespread the phenomenon is and in what contexts it is used. It will be of great interest to determine what advantage is conferred by evolving (or retaining) this baroque mechanism of the mRNA maturation.

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5. REFERENCES

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Johnson, P. J., Kooter, J. M. and Borst, P. (1987) Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene. Cell 51: 273-281. Muhich, M. L. and Boothroyd, J. C. (1988) Polycistronic transcripts in trypanosomes and their accumulation during heat shock: evidence for a precursor role in mRNA synthesis. Mol. Cell Biol. 8: 3837-3846. Tschudi, C. and Ullu, E. (1988) Polygene transcripts are precursors to calmodulin mRNAs in trypanosomes. EMBO J. 7: 455-463. Tschudi, C. and Ullu, E. (1990) Destruction of U2, U4 or U6 small nuclear RNAs blocks trans-splicing in trypanosome cells. Cell 61: 459-466. Huang, J. and van der Ploeg, L. H. T. (1991) Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans-splicing and poly (A) addition at nascent RNA transcripts from the hsp 70 locus. Mol. Cell Biol. 11: 3180-3190. Chung, H. M., Lee, M. G-S. and van der Ploeg, L.H.T. (1993) RNA polymerase I-mediated protein coding gene expression in Trypanosoma brucei. Parasit. Today 8:414-418. Zomerdijk, J. C. B. M., Ouellette, M., ten Asbroek, A. L. M. A. et al. (1990) The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei. EMBO J. 9: 2791-2801. Brown, S. D., Huang, J. and van der Ploeg, L. H. T. (1992) The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters. Mol. Cell Biol. 12: 2644-2652. Sherman, D. R., Janz, L., Hung, M. and Clayton, C. (1991) Anatomy of the parp gene promoter of Trypanosoma brucei. EMBO J. 10: 3379-3386. Pays, E., Coquelet, H., Tebabi, P. et al. (1990) Trypanosoma brucei: constitutive activity of the VSG and procyclin promoters. EMBO J. 10: 3145-3151. Ullu, E., Matthews, K. R. and Tschudi, C. (1993) Temporal order of RNA process reactions in trypanosomes: rapid trans-splicing precedes polyadenylation of newly-synthesized tubulin transcripts. Mol. Cell Biol. 13: 720-725. Spieth, J., Lea, K., Brooke, B. and Blumenthal, T. (1993) Operons in C. eleoans: polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73: 521-532. Hannon, G. J., Maroney, P. A., Ayers, D. G., Shambaugh, J. D. and Nilsen, T. W. (1990). Transcription of a nematode trans-spliced leader RNA requires internal elements for both initiation and 3' end formation. EMBO J. 9: 1915-1921. Maroney, P. A., Hannon, G. J. and Nilsen, T. W. (1990) Transcription and cap trimethylation of a nematode spliced leader RNA in a cell free system. Proc. Natl. Acad. Sci. USA 87: 709-713. Ach, R. A. and Weiner, A. M. (1987) The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation. Mol. Cell Biol. 7: 2070-2079. Parry, H. D., Scherly, D. and Mattaj, I. W. (1989) 'Snurpogenesis': the transcription and assembly of U snRNP components. Trends Biochem. Sci. 14: 15-19. Agabian, N. (1990) Trans-splicing of nuclear pre-mRNAs. Cell 61: 1157-1160. Blumenthal, T. and Thomas, J. (1988) Cis and trans-mRNA splicing in C. elegans. Trends Genet. 4: 305-308. Borst, P. (1986) Discontinuous transcription and antigenic variation in trypanosomes. Annu. Rev. Biochem. 55: 701-732. Nilsen, T. W. (1993) Trans-splicing of nematode pre-messenger RNA Annu. Rev. Microbiol. 47:413-440. Krause, M. and Hirsh, D. (1987) A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49: 753-761. Rajkovic, A., Davis, R. E., Simonsen, J. N. and Rottman, F. M. (1990) A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni. Proc. Natl. Acad. Sci. USA 87: 8879-8883.

1. MOLECULAR BIOLOGY OF PROTOZOAN AND HELMINTH PARASITES 17 23. Tessier, L.-H., Keller, M., Chan, R., Fournier, R., Weil, J. H. and Imbault, P. (1991) Short leader sequences may be transferred from small RNAs to pre-mature mRNAs by transsplicing in Euglena EMBO J. 10: 2621-2625. 24. Murphy, W. J., Watkins, K. P. and Agabian, N. (1986) Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans-splicing. Cell 47:517-525. 25. Sutton, R. and Boothroyd, J. C. (1986) Evidence for trans-splicing in trypanosomes. Cell 47: 527-535. 26. Luhrmann, R. (1988) snRNP proteins. In: Small Nuclear Ribonucleoprotein Particles (ed. Birnstiel, M. L.) Springer-Verlag, pp. 71-99. 27. Mattaj, I. W. (1988) U snRNP assembly and transport. In: Small Nuclear Ribonucleoprotein Particles (ed. Birnstiel, M. L.) Springer-Verlag, pp. 100-114. 28. Bruzik, J. P., van Doren, K., Hirsh, D. and Steitz, J. A. (1988) Trans-splicing involves a novel form of small ribonucleoprotein particles. Nature 335: 559-562. 29. Maroney, P. A., Hannon, G. J. Denker, J. A. and Nilsen, T. W. (1990) The nematode spliced leader RNA participates in trans-splicing as an Sm snRNP. EMBO J. 9: 3667-3673. 30. Bangs, J. D., Crain, P. F., Hashizume, T., McCloskey, J. A. and Boothroyd, J. C. (1992) Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J. Biol. Chem. 267: 9805-9815. 31. Palfi, Z., Giinzl, A., Cross, M. and Bindereif, A. (1991) Affinity purification of Trypanosoma brucei snRNPs reveals common and specific protein components. Proc. Natl. Acad. Sci. USA 88: 9097-9101. 32. Sharp, P. A. (1987) Trans-splicing: variation on a familiar theme? Cell 50: 147-148. 33. Laird, P. (1989) Trans-splicing in trypanosomes--archaism or adaptation? Trends Genet. 5: 204-208. 34. Hannon, G. J., Maroney, P. A., Denker, J. A. and Nilsen, T. W. (1990) Trans-splicing of nematode pre-messenger RNA in vitro. Cell 61: 1247-1255. 35. Hannon, G. J., Maroney, P. A., Yu, Y.-T., Hannon, G. E. and Nilsen, T. W. (1992) Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing interacts with U6 snRNA. Science 258: 1775-1780. 36. Maroney, P. A., Hannon, G. J., Shambaugh, J. D. and Nilsen, T. W. (1991) Intramolecular base pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro. EMBO J. 10: 3869-3875. 37. Hausner, T.-P., Giglio, L. M. and Weiner, A. M. (1990) Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev. 4: 2146-2156. 38. Watkins, K. P. and Agabian, N. (1991) In vivo UV cross-linking of U snRNAs that participate in trypanosome trans-splicing. Genes Dev. 5: 1859-1869. 39. Moore, M. J., Query, C. C. and Sharp, P. A. (1993) Splicing of precursors to messenger RNAs by the spiceosome. In: The R N A World (eds Gesterland, R. and Adkins, S.), Cold Spring Harbor Press: Cold Spring Harbor, NY, pp. 303-358. 40. Nilsen, T. W. (1994) RNA-RNA interactions in the spliceosome: Unraveling the ties that bind. Cell 78: 1-4. 41. Mottram, J., Perry, K. L., Lizardi, P. M., Luhrmann, R., Agabian, N. and Nelson, R. G. (1989) Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp, brucei: identification of the U2, U4 and U6 RNA analogs. Mol. Cell. Biol. 9: 1212-1223. 42. Tschudi, C., Richards, F. F. and Ullu, E. (1986) The U2 RNA analogue of Trypanosoma brucei gambiense: implications for splicing mechanisms in trypanosomes. Nucleic Acids Res. 14: 8893-8903. 43. Bektesh, S. L., van Doren, K. V. and Hirsh, D. (1988) Presence of the Caenorhabditis elegans spliced leader on different mRNAs and in different genera of nematodes. Genes Dev. 2: 1277-1283. 44. Stuart, K. (1993) RNA editing in trypanosomatid mitochondria. Annu. Rev. Microbiol. 45: 327-344. 45. Pollard, V. W., Rohrer, S. P. Michelotti, E. F. et al. (1990) Organization of minicircle genes for guide RNAs in Trypanosoma brueei. Cell 63: 783-790.

2

Carbohydrate and Energy Metabolism in Aerobic Protozoa FRED R. OPPERDOES International Institute of Cellular and Molecular Pathology and University of Louvain, Brussels, Belgium

SUMMARY The Trypanosomatidae are characterized by a high flexibility of their energy metabolism. They contain a single mitochondrion at all developmental stages of which the contribution to the overall ATP generation varies significantly from one member of the trypanosomatid family to another and from one life-cycle stage to another. As a consequence the contribution of carbohydrate catabolism to the overall energy generation is also highly variable. The extreme is encountered in the bloodstream forms of some African trypanosomes where cytochromes and tricarboxylic acid (TCA) cycle enzymes are absent and glycolysis is the sole source of metabolic ATP; these organisms have a very active glycolytic pathway. In all the Trypanosomatidae studied, glycolysis takes place via the Embden-Meyerhoff pathway of which the early enzymes are sequestered inside a subcellular organelle: the glycosome. At present, the order Kinetoplastida, to which the Trypanosomatidae belong, is the only known group of living organisms where this kind of compartmentation has been found. The glycosome is a member of the family of microbodies to which also belong the peroxisomes, present in almost all other eukaryotic cells, and the glyoxysomes, typical of germinating plants. The first part of this chapter discusses the African trypanosome Trypanosoma brucei and, where appropriate, other species of the Trypanosoma, and Leishmania genera will be dealt with. Data about other members of the Trypanosomatidae, such as Crithidia sp., Herpetomonas sp., Leptomonas sp. and Phytomonas sp. is highly fragmented and incomplete and therefore only dealt with where appropriate. Biochemistry and Molecular Biology of Parasites

Copyright 9 1995 Academic Press Ltd All rights of reproduction in any form reserved

ISBN 0-12-473345-X

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The second part of this chapter deals with the carbohydrate metabolism of the malaria parasite Plasmodium. Virtually nothing is known about the carbohydrate metabolism of the stages of the life cycle other than the one that infects the red blood cell. Even for this stage information on glycolysis and mitochondrial metabolism is still highly fragmented. Energy generation occurs predominantly or exclusively through fermentation of glucose with the production of lactate as the major end-product. Little is known about the role and properties of the individual enzymes involved in carbohydrate metabolism, mainly because of difficulties in obtaining sufficient parasite material free of host cell contamination. Rapid progress is now being made through the molecular cloning of several of the glycolytic enzymes.

1. THE TRYPANOSOMATIDAE 1.1. Substrates and End-products of Metabolism 1.1.1. African trypanosomes The African trypanosomes go through a complex life cycle comprising, in the vertebrate host, long slender, intermediate and short stumpy bloodstream forms, in the midgut of the tsetse fly the procyclic trypomastigote stage and in the insect's salivary glands and proboscis the epimastigote and metacyclic stages. No comprehensive study of carbohydrate metabolism through each stage of the life cycle of any one species has been made so far and little is known about the metabolism of the epimastigote and metacyclic stages. Also little is known about the metabolism of African trypanosomes other than the extensively studied T. brucei. 1.1.1.1. Vertebrate stages. All bloodstream stages (long slender, intermediate and short stumpy) of T. brucei actively catabolize glucose, fructose, mannose and glycerol. Due to the absence of a mitochondrial TCA cycle and a functional respiratory chain, the long slender forms are incapable of oxidizing amino acids or fatty acids. Short-stumpy stages are able to utilize o~-ketoglutarate in addition to glucose, fructose and glycerol (1). Bloodstream forms contain neither carbohydrate stores nor other energy reserves. Thus, depletion of exogenous substrate leads to a rapid drop of ATP levels and loss of motility. Under aerobic conditions the long slender forms metabolize glucose quantitatively to pyruvate. Trace amounts of CO 2 and sometimes glycerol have been reported as end products. However, CO 2 may result from a spontaneous decarboxylation of pyruvate, whereas the glycerol most likely results from a partial anaerobiosis of the cells during culture or incubation. Pyruvate cannot be converted into lactate because of the absence of lactate dehydrogenase; enzymatic decarboxylation of pyruvate does not occur in these forms due to the absence of pyruvate decarboxylase. In the short stumpy form pyruvate is further metabolized because a mitochondrial pyruvate decarboxylase is derepressed. In pleomorphic T. rhodesiense, comprising a

2. METABOLISM IN AEROBIC PROTOZOA

21

high percentage of stumpy forms, the major end-products of glucose metabolism are pyruvate (75%), glycerol (5%), acetate (9%), succinate (1%) and CO2 (3%). Reoxidation of glycolytically produced NADH in the glycosome is mediated through a glycerol-3-phosphate-dihydroxyacetone-phosphate cycle comprising the glycosomal NAD-linked glycerol-3-phosphate dehydrogenase and the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase-oxidase complex. The terminal oxidase reduces molecular oxygen to water without H20 2 as intermediate. This reaction does not involve cytochromes and is insensitive to the classical inhibitors of mitochondrial respiration, such as cyanide and antimycin (Fig. 2.1). Bloodstream-form trypanosomes do not exhibit a Pasteur effect. Under anaerobic conditions, or when oxygen consumption via the mitochondrial glycerol-3-phosphate oxidase is inhibited with salicyl hydroxamic acid (SHAM), long slender bloodstream forms continue to utilize glucose at about the same rate as under aerobic conditions. This is due to the fact that the glycerol-3-phosphate-dihydroxyacetone phosphate cycle is now inoperative, preventing the oxidation of glycerol-3-phosphate. The latter compound accumulates inside the glycosome, while glycosomal ATP is trapped by the phosphorylation of glucose and fructose-6-phosphate in the hexokinase (HK) and phosphofructokinase (PFK) reactions, respectively. Thus, anaerobiosis leads to high glycerol-3-phosphate and ADP concentrations and a low ATP concentration, and glycerol that rapidly equilibrates over biological membranes diffuses out of the glycosome. This creates in the glycosome the right conditions for a reversal of an otherwise irreversible glycerol kinase reaction by mass action, resulting in the utilization of glycerol-3-phosphate and ADP and the formation of glycerol and ATP. As a consequence, under anaerobic conditions, or with SHAM to inhibit respiration, glucose is dismutated into equimolar amounts of pyruvate and glycerol, with net synthesis of 1 molecule of ATP, per molecule of glucose consumed (2-4). Cells survive and remain motile under anaerobic conditions, although cellular ATP levels drop significantly (5). Glycerol, under anaerobic conditions, cannot serve as a substrate, because glycerol-3phosphate cannot be oxidized to dihydroxyacetone phosphate (DHAP) without molecular oxygen. At concentrations above several millimolar, glycerol becomes toxic. At these glycerol concentrations the reversal of the glycerol kinase reaction is no longer possible and glycolysis comes to a halt, resulting in a total disappearance of cellular ATP (6). This aspect of trypanosome glycolysis has been exploited in the treatment of experimental animals infected with either T. brucei or T. rhodesiense. The administration of a combination of SHAM and glycerol has been shown to lead to an almost immediate lysis and disappearance of the parasites from the circulation (7). Permanent cures, however, were only obtained at concentrations of the compounds that were toxic to the animals. 1.1.1.2. Insect stages. Procyclic culture forms of T. rhodesiense actively metabolize glucose, fructose, mannose or glycerol, but do not oxidize other mono- or disaccharides (8). The major end-product of glucose metabolism is CO 2 (55% of glucose carbon), together with small amounts of acetate (3%) and succinate (4%). For T. brucei procyclics 17% of the glucose carbon was recovered as succinate and 8% as alanine (9). The production of CO2 was not measured. Proline and the tricarboxylic acid cycle intermediates ~-ketoglutarate and succinate also support respiration in T. rhodesiense.

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1 h) in the presence of cycloheximide, but amino acid sequences have not yet been established. AdoMet decarboxylase is the second key enzyme in polyamine biosynthesis. In mammalian cells it is activated by putrescine and is also characterized by the presence of a PEST sequence and a short half-life (13). Trypanosome AdoMet decarboxylase, like ODC, has a long ( > 6 h) half-life as shown by in vitro incubation studies with cycloheximide using procyclic and bloodstream trypomastigotes (Bacchi, unpublished results). The crude enzyme is only 50% stimulated by 1 mM putrescine (22) but in the partly purified state it is stimulated 8-fold by 2mM putrescine (23). Trypanosome AdoMet decarboxylase, like other eukaryotic counterparts, contains a covalently

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bound pyruvate as cofactor, but its subunit composition may differ from that of other eukaryotes since antiserum against human AdoMet decarboxylase did not cross react with the partly purified trypanosome enzyme (23). Although the long half-lives of ODC and AdoMet decarboxylase in trypanosomes indicate lack of control of polyamine biosynthesis, there is evidence for some regulation. During the growth cycle of trypomastigotes in culture, a rapid rise in putrescine levels in early log phase is followed by an increase in spermidine levels and an abrupt fall in putrescine (24). These events parallel the rapid induction of ODC in log phase of procyclic forms (14) and are consistent with putrescine activation of AdoMet decarboxylase. Although the half-life data of ODC and AdoMet decarboxylase are not known for many protozoa, there is evidence for some regulation of polyamine biosynthesis during the life cycle. Plasmodium spp. undergo dramatic changes in polyamine content during the life cycle and activities of the ODC and AdoMet decarboxylase peak during the early trophozoite stage (25). In addition, bis(benzyl)polyamine analogs inhibit growth of P. falciparum in vitro and P. berghei in vivo (26). These agents are debenzylated on entry into mammalian cells and repress ODC and AdoMet decarboxylase activities (27). Depletion of polyamines by the bis(benzyl)polyamines and ODC inhibitors prevents transformation of trophozoites to schizonts (25, 27-29). Similarly, inhibition of growth with bis(benzyl)polyamines has been observed for Leishmania (30). These observations strongly suggest that enzyme product interactions have a role in regulation of polyamine biosynthesis in Plasmodium spp. and Leishmania spp. However, L. donovani ODC, unlike the mammalian enzyme, is not negatively regulated by polyamines (16).

2.2. Trypanothione Metabolism A unique feature of polyamine metabolism in trypanosomatids is the presence of a spermidine-containing peptide, NX,N8-bis(glutathionyl)spermidine (31). This unusual peptide is present only in kinetoplastid protozoa. Although E. coli contains N xglutathionylspermidine (32), it does not synthesize the bisglutathionyl conjugate. It is synthesized by addition of glutathionyl residues to the terminal amines of spermidine by a specific synthase (Fig. 7.1). Since trypanosomes lack catalase, glutathione peroxidase and glutathione reductase, trypanothione functions to detoxify free radicals (O2, OH) when the parasites are under oxidant stress (33) (see Chapter 9). Reduced trypanothione is used by the cell in the non-enzymatic reduction of oxidized glutathione. Trypanothione, in turn, is reduced by a specific FAD-containing trypanothione reductase which does not act on glutathione but exhibits 41% sequence identity with mammalian glutathione reductase (34,35). The most notable differences are the replacement of three arginine residues of the glutathione reductase (residues 37, 28 and 347) by neutral residues in trypanothione reductase (34). In human glutathione reductase the arginine-37 and arginine-347 residues form an important ionic interaction with the ~-carboxylate groups of glycine and glutamyl residues. Three acidic residues present in the trypanothione reductase active site (Glu-18, Asp-ll2 and Asp-ll6) have no counterpart in the glutathione reductase and may interact analogously with the positively charged spermidine moiety of trypanothione (34). This novel molecule and its dependence on the polyamine pathway make this a likely target for chemotherapy: its synthesis is depressed during exposure to the irreversible ODC inhibitor D L -

124

C.J. BACCHI AND N. YARLETT

~-difluoromethylornithine (DFMO) (36) and its thiol groups form stable bonds with the arsenic moieties of the trypanocide Melarsoprol, rendering it incapable of undergoing redox reactions and turning the adduct into an inhibitor of trypanothione reductase (37). In addition, trypanothione reductase from T. cruzi can reduce naphthoquinone and nitrofuran derivatives to auto-oxidizable products which in the presence of air, generate reactive oxygen species that kill the parasite (38) (see Chapter 9). Arsenical-refractory clinical isolates of T.b. rhodesiense are resistant to lysis in the presence of pharmacologically achievable blood levels of arsenical drugs whereas clinically sensitive isolates are destroyed by 1-10pM concentrations of drug. Trypanothione levels in refractory isolates decrease minimally during drug exposure (10 pM for 3 h), whereas free trypanothione is almost undetectable in sensitive strains after exposure to a lower dose (1 pM for 2 h) (39). This study and another, which reveals the absence of an adenosine transporter in arsenical-resistant trypanosomes (40), suggest that the uptake of arsenical drugs is significantly reduced in refractory trypanosome isolates.

2.3. Arginine Dihydrolase Pathway in Anaerobic Protozoa Polyamine biosynthesis in Giardia intestinalis, Trichomonas vaginalis and Tritrichomonas foetus is distinctly different from that occurring in aerobic protozoa. In these parasites arginine is the precursor of polyamine biosynthesis via the arginine dihydrolase pathway and ATP is also produced from this pathway (Fig. 7.1, and Chapter 5). The first enzyme of the pathway is arginine deiminase (EC 3.5.3.6) which is responsible for the liberation of ammonia from arginine with the resultant formation of citrulline. Citrulline undergoes phosphorolysis by catabolic ornithine carbamoyl transferase (carbamoyl phosphate: L-ornithine carbamoyl transferase, EC 2.1.3.3) to ornithine with the loss of the guanidine carbon atom as carbamoyl phosphate. Some anaerobic protozoa do not synthesize the pyrimidine ring hence the carbamoyl phosphate is not used for this purpose. Instead carbamoyl phosphate is broken down by a catabolic carbamate kinase (ATP: carbamate phosphotransferase, EC 2.7.2.2) to bicarbonate and ammonia with the concomitant phosphorylation of a molecule of ADP to ATP (Fig. 7.1). The ornithine formed is decarboxylated by ODC to form putrescine. The enzymes of this pathway, have been found in T. vaginalis (6), T. foetus (41) and G. intestinalis (42). The importance of this pathway is indicated by the rapid depletion of arginine from the growth medium and the large quantities of putrescine which result both in vitro and in vivo (12). This pathway is also assumed to be present in rumen ciliates. It has been reported that ciliates of the species Entodinium, Diplodinium, Dasytricha and Isotricha produce citrulline from arginine, ornithine from citrulline, putrescine and proline from ornithine and ~-aminovalerate from proline (43). The importance of the pathway is consistent with putrescine as the major polyamine in these species also (Yarlett, Lloyd, Williams and Ellis, unpublished). The presence of the dihydrolase pathway has not been demonstrated in Entamoeba, although this organism also produces putrescine as the major polyamine. The differences in putrescine biosynthesis between aerobic and anaerobic protozoa may be a consequence of the absence of mitochondria in the latter and may be linked to the need for alternative ATP-generating processes. This pathway has the advantage of producing a non-acidic end-product (putrescine), whereas the other major end-

7. POLYANIYE NEXABOLISN

125

products (acetate, lactate and butyrate) are acidic. Support for the idea that high concentrations of putrescine may be beneficial to organisms growing under anaerobic conditions is highlighted by the finding that plant cells, when exposed to anoxic conditions also produce putrescine as the major polyamine. Spermine and spermidine biosynthesis are inhibited under these conditions (44). It appears though that T. vaginalis is unaffected by high concentrations of spermine (1) and is capable of growth in human semen (45) which contains in excess of 10mM spermine (46).

2.4. AdoMet Metabolism in African Trypanosomes AdoMet is a critical branch point metabolite. In its decarboxylated form, it serves as aminopropyl group donor in polyamine synthesis; as AdoMet it is the methyl group donor for most transmethylation reactions (Fig. 7.1). In eukaryotic cells AdoMet decarboxylase is activated by putrescine and is the rate-limiting step of polyamine biosynthesis. Since production of dc-AdoMet is irreversible (13) regulation of AdoMet decarboxylase by putrescine determines the overall rate of polyamine synthesis. Intracellular AdoMet and dc-AdoMet increase in trypanosomes 70- and > 1000-fold after DFMO treatment in vivo (47, 48). In comparison, mammalian cells treated with DFMO barely double their AdoMet content, and increase dc-AdoMet only 500-fold (49, 50). The large increase in trypanosome AdoMet and consequent > 1000-fold rise in dc-AdoMet from almost undetectable levels, results in a combined intracellular pool of 5 m u - - a b o u t 1400 times that of normal (47). The ratio of AdoMet to its demethylated product, S-adenosylhomocysteine (AdoHcy), indicates the relative potential of the cell to methylate molecules (methylation index) (51). In bloodform trypanosomes exposed to DFMO for 36 h the methylation index also becomes elevated from a normal level of 6.5 to 114 (47). The kinetics of the AdoMet synthetase appears to favor this increase since the trypanosome enzyme has a high K i for AdoMet (240 #M) as compared to mammalian enzymes (2-40 #M) (52). Disruption of AdoMet metabolism is a key element also in the action of 5'-{[(Z)-5amino-2-butenyl]methylamino)5'-deoxyadenosine (MDL73811), an AdoMet decarboxylase inhibitor which is a highly effective trypanocide (53). Treatment of trypanosomes with MDL73811 caused a 20-fold increase in the trypanosome AdoMet level after only 1 h of exposure. In comparison, AdoMet levels in cultured mammalian cells rose only 1.5-fold after similar treatment with MDL73811 (48). This analog is rapidly concentrated by trypanosomes through the purine transport system (54). In addition to elevated AdoMet levels, trypanosomes treated with DFMO in vivo experience a six-fold increase in the activity of protein methylase II but not I or III (55). Protein methylase II specifically methylates carboxyl groups of aspartate and glutamate residues. In mammalian cells, histone methylation may be involved in the condensation of euchromatin to heterochromatin prior to mitosis (56-58), and may therefore have a role in gene expression. Aspartate- and glutamate-rich histones have also been characterized from both T. b. brucei and Crithidiafasciculata making these likely substrates for protein methylase II activity (59, 60). These observations indicate that synthesis of AdoMet in African trypanosomes is unregulated as compared to the mammalian host and that trypanosomes may therefore be more sensitive to imbalances in the AdoMet :AdoHcy ratio (methylation index). Morphological alterations (long slender to stumpy transformation) in African

126

C.J. BACCHI AND N. YARLETT

trypanosomes during DFMO treatment and accompanying changes in mitochondrial structure attributed to polyamine depletion (11, 61) suggest that alterations in methylation patterns may have a significant role in the life cycle. Penketh et al. (62) found that methylating agents such as streptozotocin and procarbazine were specifically trypanocidal and caused synchronous differentiation of blood forms into short stumpy forms.

3. POLYAMINE METABOLISM IN HELMINTHS Most data concerning polyamine synthesis and content of parasitic helminths concern adult worms. Helminths differ from the majority of eukaryotes in that they contain spermidine and spermine and lesser or undetectable amounts of putrescine (Table 7.1) (2, 63-66). Cadaverine has also been detected in some helminths (2), although an active lysine decarboxylase has not been conclusively demonstrated, and cadaverine has no known function in eukaryotic cells (67). The intracellular concentration of spermidine and spermine in helminths is similar to that of vertebrate cells. There are indications that helminths lack polyamine biosynthetic enzymes (ODC, ADC) and may be dependent on uptake from the host (64-66). However, helminths are capable of interconversion of polyamines taken up from the host, suggesting the involvement of a polyamine salvage pathway (64). Spermine is converted into N-acetylspermine and then spermidine via the combined actions of N-acetylspermine transferase and polyamine oxidase (Fig. 7.1). Spermidine is then converted into N a- or N8-acetylspermidine and putrescine via the combined actions of N 1- or N8-acetylspermidine transferase and polyamine oxidase (Fig. 7.1) (64, 65). In addition, helminths can convert putrescine into N-acetylputrescine, apparently a major excretory product (64,66). Most of the ornithine decarboxylating activity present in helminths is insensitive to DFMO but is sensitive to glutamate (65). It is likely that the release of CO 2 from ornithine is due to an ornithine aminotransferase which produces glutamic semialdehyde and then glutamate via glutamate semialdehyde dehydrogenase. Glutamate is subsequently decarboxylated to 7-aminobutyric acid (65, 68). Two observations support this mechanism: (a) inhibition of the ornithine aminotransferase pathway with gabaculine (3-amino-2,3dihydrobenzoic acid) a suicide inhibitor, abolishes CO 2 release from ornithine (64); (b) Onchocerca volvulus, a filariid nematode which lacks the ornithine aminotransferase pathway, does not liberate CO 2 from ornithine (64). Although these studies indicate a lack of polyamine biosynthesis in helminths, it is important to note that larval stages have not been examined and that the potential for stage-specific activation should not be ignored.

4. CONSIDERATIONS IN THE INHIBITION OF POLYAMINE METABOLISM IN PARASITE CHEMOTHERAPY The overall pathways of polyamine metabolism in pathogenic protozoa and helminths seem to be sufficiently different from the mammalian host to afford multiple opportunities for drug intervention. The important features are: the long half-life of ODC in kinetoplastids coupled with poor polyamine transport and the apparently unregulated

7. POLYAMINE METABOLISM

127

synthesis of AdoMet. Some anaerobic protozoa such as Trichomonas, Giardia and Entamoeba spp. also excrete large quantities of putrescine, and may lack polyamine biosynthetic capabilities. These parasites rely in part on ATP production via the arginine dihydrolase pathway and may be particularly susceptible to arginine analogs or similar agents interfering with enzymes of the dihydrolase pathway. There is evidence that T. cruzi possesses arginine decarboxylase activity, which is sensitive to inhibition by difluoromethylarginine (9). In addition difluoromethylarginine is capable of reducing the infectivity of T. cruzi to mammalian cells (8, 10), whereas ODC inhibitors have no effect on infectivity (8). Specific inhibition of infectivity results from selective impairment of amastigote division without affecting the capacity of the invading trypomastigotes to transform into the replicative amastigote form (10). Thus, this pathway is a potential target for selective chemotherapy, both because mammalian cells lack arginine decarboxylase and because the enzyme appears to be critical for parasite invasion of host cells and amastigote multiplication (10). As noted in Chapter 6, trypanosomes and the anaerobic protozoa are purine auxotrophs with significant capacity to transport (69-71), interconvert and salvage purines (72; see Chapter 6). Although chemotherapy based on purine salvage of these protozoa is now being developed (72), there is evidence that critical targets within AdoMet metabolism may also be vulnerable. AdoMet synthetase in kinetoplastids exhibits a high Ki for product inhibition, as well as a broad methionine requirement (52). The purine requirement for this enzyme is unknown and it is likely that an AdoMet analog based on purine or methionine could affect both polyamine and methylation reactions. In trypanosomatids, the salvage enzyme methylthioadenosine (MTA) phosphorylase has been found to have a broad substrate requirement (Fig. 7.1) (73). In African trypanosomes this enzyme also cleaves 5'-halo- and ethylthioribose derivatives of MTA to produce adenine plus the resulting ribose analog (74). The latter would normally be recycled to methionine (5). These agents are trypanocidal and their effects can be reversed with methionine or ketomethylthiobutyrate, an intermediate in the conversion of methylthioribose into methionine (74). A critical detoxifying and salvage enzyme within the transmethylation pathway is AdoHcy hydrolase. This intermediate is highly toxic to cells and is rapidly hydrolyzed to adenine and homocysteine (75). African trypanosomes have high levels of AdoHcy hydrolase (47) and are susceptible to the AdoHcy analog Neplanocin A (75). Although this analog is toxic to mammalian cells, the range of substrate specificity for this enzyme is unknown and should be pursued for both trypanosomes and other protozoa.

5. REFERENCES 1. Yarlett, N. and Bacchi, C. J. (1988) Effects of DL-~-difluoromethylornithine on polyamine synthesis and interconversion in Trichomonas vaginalis grown in a semi-defined medium. Mol. Biochem. Parasitol. 31: 1-10. 2. Gordon, R., Cornect, M., Waiters, B. M., Hall, D. E. and Brosnan, M. E. (1989) Polyamine synthesis by the mermithid nematode Romanomermis culicivorax. J. Nematol. 21: 81-86. 3. Pegg, A. E. (1986) Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J. 234: 249-262. 4. Pegg, A. E. and McCann, P. P. (1988) Polyamine metabolism and function in mammalian cells and protozoans. ISI Atlas of Science Biochemistry. ICI Publishers, Cleveland, Ohio, pp. 11-18.

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5. Porter, C. W. and Sufrin, J. R. (1986) Interference with polyamine biosynthesis and/or function by analogs of polyamines or methionine as a potential anticancer chemotherapeutic strategy. Anticancer Res. 6: 525-542. 6. Linstead, D. and Cranshaw, M. A. (1983) The pathway of arginine catabolism in the parasitic flagellate Trichomonas vaoinalis. Mol. Biochem. Parasitol. 8: 241-252. 7. McCann, P. P., Bacchi, C. J., Bitonti, A. J., Kierszenbaum, F. and Sjoerdsma, A. (1988) In: Progress in Polyamine Research (eds Zappia, V. and Pegg, A. E.), Plenum Press, New York, pp. 727-736. 8. Kierszenbaum, F., Wirth, J. J., McCann, P. P. and Sjoerdsma, A. (1987) Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells. Proc. Natl. Acad. Sci. USA 84: 4278-4282. 9. Majumder, S., Wirth, J. J., Bitonti, A. J., McCann, P. P. and Kierszenbaum, F. (1992) Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi. J. Parasitol. 78: 371-374. 10. Yakubu, M. A., Basso, B. and Kierszenbaum, F. (1992) DL-~-difluoromethylarginine inhibits intracellular Trypanosoma cruzi multiplication by affecting cell division but not trypomastigote-amastigote transformation. J. Parasitol. 78: 414-419. 11. Bacchi, C. J. and McCann, P. P. (1987) In: Inhibition of Polyamine Metabolism (eds McCann, P. P., Pegg, A. E. and Sjoerdsma, A.) Academic Press, New York, pp. 317-344. 12. Yarlett, N. and Bacchi, C. J. (1991) In: Biochemical Protozoolooy; Basis for Dru9 Desion (eds Coombs, G. H. and North, M. J.), Taylor & Francis, London, pp. 458-468. 13. Pegg, A. E. (1988) Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48: 759-774. 14. Bacchi, C. J., Garofalo, J., Santana, A., Hannan, J. C., Bitonti, A. J. and McCann, P. P. (1989) Trypanosoma brucei brucei: Regulation of ornithine decarboxylase in procyclic forms of trypomastigotes. Exp. Parasitol. 68: 392-402. 15. Assaraf, Y. G., Kahana, C., Spira, D. T. and Bachrach, U. (1988) Plasmodium falciparum: Purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis. Exp. Parasitol. 67: 20-30. 16. Hanson, S., Adelman, J. and Ullman, B. (1992) Amplification and molecular cloning of the ornithine decarboxylase gene of Leishmania donovani. J. Biol. Chem. 267: 2350-2359. 17. Yarlett, N., Goldberg, B., Moharrami, M. A. and Bacchi, C. J. (1992) Inhibition of Trichomonas vaginalis ornithine decarboxylase by amino acid analogs. Biochem. Pharmacol. 44: 243-250. 18. Phillips, M. A., Cotfino, P. and Wang, C. C. (1987) Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Mol. Biochem. Parasitol. 22: 9-17. 19. Ghoda, L., Phillips, M. A., Bass, K. E., Wang, C. C. and Coffino, P. (1990) Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation. J. Biol. Chem. 254: 11823-11826. 20. Rogers, S., Wells, R. and Rechsteiner, M. (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234: 364-368. 21. Coons, T., Hanson, S., Bitonti, A. J., McCann, P. P. and Ullman, B. (1990) Alphadifluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity. Mol. Biochem. Parasitol. 39: 77-90. 22. Bitonti, A. J., Bacchi, C. J., McCann, P. P. and Sjoerdsma, A. (1986) Uptake of ~-difluoromethylornithine by Trypanosoma brucei brucei. Biochem. Pharmacol. 35: 351-354. 23. Tekwani, B. L., Bacchi, C. J. and Pegg, A. E. (1992) Putrescine activated S-adenosylmethionine decarboxylase from Trypanosoma brucei brucei. Mol. Cell. Biochem. 117: 53-61. 24. Bacchi, C. J., Lipschik, G. Y. and Nathan, H. C. (1977) Polyamines in trypanosomatids. J. Bacteriol. 131: 657-661. 25. Assaraf, Y. G., Golenser, J., Spira, D. T. and Bachrach, U. (1984) Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malarial parasite Plasmodium falciparum. Biochem. J. 222: 815-819. 26. Bitonti, A. J., Dumont, J. A., Bush, T. L. et al. (1989) Bis(benzyl) polyamine analogs inhibit the growth of chloroquine resistant human malaria parasites (Plasmodium falciparum) in vitro

7. POLYAMINE METABOLISM

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

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and in combination with a-difluoromethylornithine cure malaria. Proc. Nat. Acad. Sci. USA. 86: 651-655. Bitonti, A. J., McCann, P. P. and Sjoerdsma, A. (1991) In: Biochemical Protozoology (eds Coombs, G. and North, M.) Taylor & Francis, London, pp. 517-523. Bitonti, A. J., McCann, P. P. and Sjoerdsma, A. (1987) Plasmodium falciparum and Plasmodium berghei: Effect of ornithine decarboxylase inhibitors on Erythrocytic schizooony. Exp. Parasitol. 64, 237-243. Wright, P. S., Byers, T. L., Cross-Doersen, D. E., McCann, P. P. and Bitonti, A. J. (1991) Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparuminfected erythrocytes: Growth inhibition in vitro. Biochem. Pharmacol. 41: 1713-1718. Baumann, R. J., Hanson, W. L., McCann, P. P., Sjoerdsma, A. and Bitonti, A. J. (1990) Suppression of both antimony-susceptible and antimony-resistant Leishmania donovani by a bis(benzyl) polyamine analog. Antimicrob. Agents Chemother. 34: 722-727. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. and Cerami, A. (1985) Trypanothione: a novel bis(glutathionyl) spermidine cofactor for glutathione reductase in trypanosomatids. Science 227: 1485-1487. Tabor, H. and Tabor, C. W. (1975) Isolation, characterization, and turnover of glutathionylspermidine from Escherichia coll. J. Biol. Chem. 250: 2648-2654. Fairlamb, A. H. and Cerami, A. (1992) Metabolism and functions of trypanothione in the kinetoplastida. Annu. Rev. Microbiol. 46: 695-729. Henderson, G. B., Murgolo, N. J., Kuriyan, J. et al. (1991) Engineering the substrate-specificity of glutathione reductase toward that of trypanothionine reduction. Proc. Natl. Acad. Sci. USA. 88: 8769-8773. Hunter, W. N., Smith, K., Derewenda, Z. et al. (1990) Initiating a crystallographic study of trypanothionine reductase. J. Mol. Biol. 216: 235-237. Fairlamb, A. H., Henderson, G. B., Bacchi, C. J. and Cerami, A. (1987) In vivo effects of difluoromethylornithine on trypanothionine and polyamine levels in bloodstream forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 24: 185-191. Fairlamb, A. H., Henderson, G. B. and Cerami, A. (1989) Trypanothione is the primary target for arsenical drugs against African trypanosomes. Proc. Natl. Acad. Sci. USA 86: 2607-2611. Henderson, G. B., Ulrich, P., Fairlamb, A. H. et al. (1988) 'Subversive' substrates for the enzyme trypanothionine disulfide reductase: alternative approach to chemotherapy of Chagas' disease. Proc. Natl. Acad. Sci. USA. 85: 5374-5378. Yarlett, N., Goldberg, B., Nathan, H. C., Garofalo, J. and Bacchi, C. J. (1991) Differential sensitivity of Trypanosoma brucei rhodesiense isolates to in vitro lysis by arsenicals. Exp. Parasitol. 72:205-215. Carter, N. and Fairlamb, A. H. (1993). Arsenical-resistant trypanosomes lack an unusual adenosine transporter. Nature 361: 173-176. Yarlett, N., Lindmark, D. G., Goldberg, B., Moharrami, M. A. and Bacchi, C. J. (1994) Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Trichomonas foetus. J. Euk. Microbiol. 41: 554-559. Schofield, P. J. and Edwards, M. R. (1991) In: Biochemical Protozoology (eds Coombs, G and North, M.), Taylor and Francis, London, pp. 102-112. Onodera, R., Yamaguchi, Y. and Morimoto, S. (1983) Metabolism of arginine, citrulline, ornithine and proline by starved rumen ciliate protozoa. Agric. Biol. Chem. 47: 821-828. Reggiani, R., Hochkoeppler, A. and Bertani, A. (1989) Polyamines and anaerobic elongation of rice coleoptile. Plant Cell Physiol. 30: 893-898. Daly, J. J., Sherman, J. K., Gren, L. and Hostetler, T. L. (1989) Survival of Trichomonas vaginalis in human semen. Genitourin. Med. 65: 106-108. Williams-Ashman, H. G. (1989) In: The Physiology of Polyamines Vol. 1 (eds. Bachrach, U. and Heimer, Y. M.), CRC Press, Boca Raton, pp. 3-20. Yarlett, N. and Bacchi, C. J. (1988) Effect of DL-a-difluoromethylornithine on methionine cycle intermediates in Trypanosoma brucei brucei. Mol. Biochem. Parasitol. 27: 1-10. Byers, T. L., Bush, T. L., McCann, P. P. and Bitonti, A. J. (1991) Antitrypanosomal effects of polyamine biosynthesis inhibitors correlate with increases in Trypanosoma brucei brucei S-adenosyl-L-methionine. Biochem. J. 274: 527-533.

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49. Mamont, P. S., Danzin, C., Wagner, J., Siat, M., Joder-Ohlenbusch, A. M. and Claverie, N. (1982) Accumulation of decarboxylated S-adenosyl-L-methionine in mammalian cells as a consequence of the inhibition of putrescine biosynthesis. Eur. J. Biochem. 123: 499-504. 50. Pegg, A. E. and McCann, P. P. (1982) Polyamine metabolism and function. Am. J. Physiol. 243 (Cell Physiol. 12): C212-C221. 51. Ueland, P. M. (1982) Pharmacological and biochemical aspects of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase. Pharmacol. Rev. 34, 223-253. 52. Yarlett, N., Garofalo, J., Goldberg, B. et al. (1993) S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei. Biochim. Biophys. Acta 1181: 68-76. 53. Bitonti, A. J., Byers, T. L., Bush, P. J. et al. (1990) Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of Sadenosylmethionione. Antimicrob. Agents Chemother. 34, 1485-1490. 54. Byers, T. L., Casara, P. and Bitonti, A. J. (1992) Uptake of the antitrypanosomal drug 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811) by the purine transport system of Trypanosoma brucei brucei. Biochem. J. 283, 755-758. 55. Yarlett, N., Quamina, A. and Bacchi, C. J. (1991) Protein methylase in Trypanosoma brucei brucei: Activities and response to DL-~t-difluoromethylornithine. J. Gen. Microbiol. 137: 717-724. 56. Tidwell, T., Allfrey, V. G. and Mirsky, A. E. (1968) The methylation of histones during regeneration of liver. J. Biol. Chem. 243: 707-715. 57. Shepherd, G. R., Hardin, J. M. and Noland, B. J. (1971) Methylation of lysine residues of histone fractions in synchronized mammalian cells. Arch. Biochem. Biophys. 143: 1-5. 58. Paik, W. K., Lee, H. W. and Morris, H. P. (1972) Protein methylases in hepatomas. Cancer Res. 32: 37-40. 59. Bender, K., Betschart, B., Schaller, J., Kaupfer, U. and Hecker, H. (1992) Structural differences between the chromatin of procyclic Trypanosoma brucei brucei and of higher eukaryotes as probed by immobilized typsin. Acta Trop. 50:169-184. 60. Duschak, V. G. and Cazzulo, J. J. (1990) The histomes of the insect trypanosomatid, Crithidia fasciculata. Biochim. Biophys. Acta. 1040: 159-166. 61. Gittin, B. F. and McCann, P. P. (1989) Physiological activation of the mitochondrion and the transformation capacity of DFMO induced intermediate and short stumpy bloodstream form trypanosomes. Am. J. Trop. Med. Hyg. 40, 487-493. 62. Penketh, P. G., Krishnamurthy, S., Divo, A. A., Patton, C. L. and Sartorelli, A. C. (1990) Methylating agents as trypanocides. J. Med. Chem. 33: 730-732. 63. Singh, R. P., Saxena, J. K., Ghatak, O. P., Wittich, R.-M. and Walter, R. D. (1989) Polyamine metabolism in Setaria cervi. Parasitol. Res. 75:311-315. 64. Walter, R. D. (1988) Polyamine metabolism of filaria and allied parasites. Parasitol. Today 4: 18-20. 65. Sharma, V., Tekawani, B. L., Saxena, J. K. et al. (1991) Polyamine metabolism in some helminth parasites. Exp. Parasitol. 72: 15-23. 66. Muller, S., Wittich, R.-M. and Walter, R. D. (1988) In: Progress in Polyamine Research (eds Zappia, V. and Pegg, A. E.), Plenum Press, New York, pp. 737-743. 67. Kallio, A. and Janne, J. (1983) Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine antimetabolites. Biochem. J. 212: 895-898. 68. Sharma, V., Katiyar, J. C., Ghatak, S. N. and Shukla, O. P. (1988) Ornithine aminotransferase of hookworm parasites. Indian J. Parasitol. 12: 237-242. 69. Jarroll, E. L. and Lindmark, D. G. (1990) Giardia metabolism. In: Giardiasis (ed. Meyer, E. A.), Elsevier, Amsterdam, pp. 61-76. 70. Wang, C. C., Verham, R., Rice, A. and Tzeng, S. (1983) Purine salvage by Trichomonasfoetus. Mol. Biochem. Parasitol. 8: 325-337. 71. Gottlieb, M. and Dwyer, D. M. (1988) In: The Biology of Parasitism (eds Englund, P. T. and Sher, A.), Alan R. Liss, New York, pp. 449-465. 72. Marr, J. J. and Docampo, R. (1986) Chemotherapy for Chagas' disease: perspective of current therapy and considerations for future research. Rev. Infect. Dis. 8: 884-903.

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73. Ghoda, L. Y., Savarese, T. M., Northup, C. H. et al. (1988) Substrate specificities of 5'-deoxy-5'-methylthioadenosine phosphorylase from Trypanosoma brucei brucei. Mol. Biochem. Parasitol. 27, 109-118. 74. Bacchi, C. J., Sufrin, J., Nathan, H. C. et al. (1991) 5'-Alkyl-substituted analogs of 5'-methylthioadenosine as trypanocides. Antimicrob. Agents Chemother. 35:1315-1320. 75. Chiang, P. K. and Miura, G. A. (1986) In: Biological Methylation and Drug Design (eds Borchardt, R. T., Creveling, C. R. and Ueland, P. M.), Humana Press, Clifton, pp. 239-252. 76. Kneifel, H., Stetter, K. O., Andreeson, J. R., Wiegel, J., Konig, H. and Schoberth, S. M. (1986) Distribution of polyamines in representative species of archaebacteria. System. Appl. Microbiol. 7: 241-245.

8

Lipid and Membrane Metabolism of the Malaria Parasite and the African Trypanosome WALLACE R. FISH Pediatric Endocrinology, State University of New York~ Health Science Center at Syracuse, Syracuse, N.Y., USA.

SUMMARY Comparison of the lipids and lipid and membrane metabolism of the malaria parasite and the African typanosome with that of their environments emphasizes the unique ways in which parasites are able to access the nutrients available, and modify them to their advantage. The differences between the salivarian trypanosomes and the Plasmodium species highlight the distinctions between situations where the host has limited access to the parasite (the erythrocyte-sequestered malaria parasite), and a totally extracellular existence (the bloodstream and insect form African trypanosome). The intraerythrocytic stages of the malaria parasite must not only accommodate the lipid requirements of both differentiation and massive multiplication, but also must prepare infective merozoites for re-invasion of erythrocytes. The African trypanosome, which produces only one daughter cell while maintaining a very rapid metabolic rate, must anticipate the stresses of temperature and nutrient changes upon entering the tsetse fly midgut. This is partially accomplished through significant alteration in the lipid composition of the bloodstream stumpy form. In both cases the continuation of the life cycle is the primary goal. Both organisms acquire host lipid, albeit through different paths, possess significant pathways to modify these lipids, and have enzymic activities, for example phospholipases or transporters with unusual properties, which are able to facilitate the process. The malaria parasite, sequestered within the host Biochemistry and Molecular Biology of Parasites

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erythrocyte, may manipulate the host membrane lipid composition and shows greater biosynthetic power than the trypanosome. Once host lipid or lipid constituents are acquired, both organisms respond in a similar fashion--modification of the gross lipid composition and, in some cases, the fatty acyl chain composition of complex lipids. In most cases it is unknown why these changes are needed. The malaria parasite may be attempting to alter the permeability of the parasitized cell to allow more facile entry of nutrients. A significant part of the African typanosomes' lipid metabolism is directed toward synthesis of not only membrane lipid, but supply of the glycolipid anchor of its unique surface coat.

1. INTRODUCTION Protozoan parasites must interact with the host at a variety of levels: the acquisition of nutrients; evasion or confusion of the host's immune response; establishment and maintenance of the infected state. This interface and the interactions are, by necessity, membrane mediated. Most parasites possess more than one life cycle stage, including those in transmission vectors, and exist at various temperatures and in different milieus; this requires significant membrane alterations. The success of parasitic organisms shows that these processes are well established. They are reflected in parasite lipid metabolism which includes the classical definition as well as 'remodeling' of host lipids and membranes. This chapter will discuss the Apicomplexan intracellular parasites Plasmodium and Toxoplasma and compare them with the extracellular African trypanosomes. The lipid and membrane metabolism of the Apicomplexa, particularly Plasmodium, has been extensively reviewed (1-4), and only phenomena of current investigation are presented. The lipid metabolism of the African trypanosomes will necessitate a more complete discussion. The reader is encouraged to supplement this information by correlating it with the discussion of glycoproteins in Chapter 11. Their location and position are largely dependent on lipid anchoring but their functions are quite specific to their structure. Their function is a result of the complementarity of lipid and glycoprotein biochemistry.

2. THE APICOMPLEXAN PARASITES In Apicomplexan parasites such as Plasmodium, Toxoplasma and Babesia lipid metabolism may be involved in invasion of host cells, concomitant parasitophorous vacuole formation, and differentiation and growth throughout the life cycle. During invasion of host cells and formation of the parasitophorous vacuole considerable membrane deformation, reformation and growth occur. The parasitophorous vacuole membrane is formed very rapidly upon invasion of the host cell through the activity of organellar (rhoptry) lipids (5). A variety of polypeptides also participate (4, 6, 7). The two processes of merozoite invasion and parasitophorous vacuole formation may be considered as continuous although distinct steps are apparent. Mikkelsen et al. (8) observed erythrocytes during, and after, invasion by P. falciparum merozoites metabolically labeled with fluorescent phospholipids (PhL). Upon invasion, lipids in the merozoite apical end

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distributed to the merozoite-erythrocyte membrane junction and into the erythrocyte membrane. After establishment of intracellular infection, the label was observed only in the parasitophorous vacuole membrane and parasite plasma membrane. It was concluded that no significant membrane metabolism had occurred; parasitophorous vacuole membrane formation involved the insertion of parasite lipid into the host erythrocyte membranes. Dluzewski et al. (9) followed invasion by P. falciparum, and parasitophorous vacuole membrane formation, of erythrocytes labeled with tagged phosphatidylethanolamine (PE). The absence of the label from both the merozoite attachment site and the region containing the intraerythrocytic parasite indicated that the parasitophorous vacuole membrane was predominantly, or wholly, of parasite origin. Only one partially characterized Plasmodium-specific lipid, a phosphoglycolipid, has been reported to be involved directly in erythrocyte invasion (10). Immunolocalization showed concentration at the merozoite apical end and the lipid was present where membrane deformation occured during erythrocyte invasion. The antibody also inhibited merozoite invasion in vitro. Other studies suggest that merozoite lipids are not the source of the parasitophorous vacuole membrane. Haldar and Uyetake (11) observed that when erythrocytes were labeled with a fluorescent lipophilic probe it was internalized upon invasion of erythrocytes by P. falciparum merozoites, but not during subsequent growth. Ward et al. (12) concluded that labeled lipids in the erythrocyte membrane are indistinguishable from those of the forming parasitophorous vacuole membrane. The deposition of fluorescent lipophilic probes in the erythrocyte membrane was followed during merozoite invasion, and the probe did not change apparent concentration in any membrane during invasion and parasitophorous vacuole membrane formation, indicating that there was no addition of exogenous lipid. Ultrastructural observations suggested that during invasion Plasmodium rhoptries extrude copious membranous material (see ref. 6 and references therein), which may provide a matrix for rhoptry proteins (13). Some rhoptry proteins bind to both erythrocyte inside-out vesicles and to vesicles prepared from phospholipids predominant in the erythrocyte membrane inner leaflet, particularly phosphatidylserine (PS) and phosphatidylinositol (PI) (14, 15). One has been shown to intercalate into the erythrocyte membrane (16). These proteins, the RHOP-H complex and SERA, are proposed to integrate differentially into the erythrocyte membrane upon invasion, causing an inward membrane expansion, to produce the parasitophorous vacuole. On balance, the evidence points to rhoptry lipids being involved, at the least, in sequestering proteins which, upon introduction into the erythrocyte membrane with or without rhoptry lipids, are directly responsible for the destabilization of the erythrocyte membrane, and subsequent parasitophorous vacuole membrane formation. Whether rhoptry lipids actually become an integral part of the parasitophorous vacuole membrane, wholly or in combination with erythrocyte lipids, is not clear. Rhoptry lipids probably combine directly with erythrocyte lipids in the parasitophorous vacuole membrane. Both lipids and polypeptides are implicated in host cell invasion by the related parasite Toxoplasma gondii (for review, see refs. 17, 18). The rhoptry lipid composition of T. gondii is unusual (19). The cholesterol (Cho) to PhL ratio is high (1.48), and phosphatidylcholine (PC) accounted for greater than 75% of the rhoptry total lipids (TL). The remaining identifiable species were PE, phosphatidic acid (PA)

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and lysolipids. Notable was the absence of PS, PI and sphingomyelin (SM), which are found in whole parasites. If this unusual rhoptry lipid composition were transferred to the parasitophorous vacuole membrane, it could participate in the binding and stabilization of vimentin-type intermediate filaments to the Toxoplasma parasitophorous vacuole membrane (20). These filaments may play a part in the aggregation of host cell organelles around the parasitophorous vacuole and, thus, could participate in supplying metabolites for biosynthesis (20). A parasite phospholipase (most likely PLA2) may participate in host cell invasion by T. 9ondii (21). The presumptive PLA 2 enhanced host cell invasion. Phospholipase has been shown to solubilize rhoptry proteins of P. falciparum presumably involved in erythrocyte invasion (22). Solubilization of internal, latent, membrane-bound rhoptry proteins by lipid degradation may be a general method in the Apicomplexa for initiating active host cell penetration and parasitophorous vacuole membrane formation after attachment.

2.1. Intracellular Growth

Once established within the erythrocyte, Apicomplexan parasites undergo multiple transformations and growth. Plasmodium species are unable to synthesize fatty acids (FA) de novo, and their ability to saturate, desaturate, elongate or shorten fatty acids is severely limited or non-existent (2, 23). The parasite must acquire much of its lipid either from the parasitized host cell or the external milieu. How does the malaria parasite provide for the tremendous increase in lipid necessary to assemble the increased membranous material? For P. falciparum, and the related parasite Babesia divergens, high density lipoproteins (HDL) are a major source of preformed lipid, in particular PC, significant amounts of which were converted into PE (24,25). A unidirectional flux of PC to the erythrocyte membrane from HDL and subsequently to the intracellular parasite was proposed for both parasites. Malaria-infected erythrocytes can acquire phospholipids (PC, PE, PS) directly from lipid vesicles; this phenomenon, which is enhanced by PhL transfer proteins, may involve exchange with the erythrocyte membrane (26). Pouvelle et al. (27) describe an organelle of parasite origin (the 'parasitophorous duct') within P. falciparum-infected erythrocytes that directly connects the erythrocyte and parasitophorous vacuole membranes. High density lipoproteins could be transported through this organelle by passive diffusion to the interior; the intraerythrocytic parasite may acquire HDL by fluid phase endocytosis. Specific antibodies against the two major HDL apolipoproteins do not localize within intracellular parasites, suggesting that lipids must be acquired from HDL directly, without endocytosis (24). However, the existence of the parasitophorous duct has been questioned (11, 12, 28). Thus, the infected erythrocyte has access to host plasma lipids, predominantly from HDL. Further, the HDL, or its lipids, must be transported from the erythrocyte membrane and through the parasitophorous vacuole membrane to the developing parasite (for review see ref. 29). The importance of this for Plasmodium is illustrated by the work of Grellier et al. (30) who showed that HDL alone supports the differentiation and multiplication of the parasite. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) or apolipoproteins do not support complete schizogony and

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subsequent erythrocyte reinvasion. Host lipid, therefore, is essential for parasite growth, differentiation and continuation of the life cycle. These lipids are remodeled by the parasite. The uptake of exogenous choline for PC synthesis is accepted (31, 32), although the nature of transport is contentious (33, 34). The disparate observations are perhaps due to variance in experimental technique: age of the erythrocytes employed; cation imbalance; reaction termination conditions; in vitro choline concentration; and innate characteristics of the two Plasmodium species examined (33, 34). It is also possible that interspecies differences in the enzymes involved in the ensuing metabolic fate of choline, for example choline kinase (35) or choline phosphotransferase (36), also influence the results, as well as interactions between the alternative pathways of PC synthesis from PE and PS. In a study of regulation of PC biosynthesis in P. knowlesi-infected erythrocytes (37), the parasite showed a comprehensive ability to modulate the pathway of PC synthesis from external choline depending on the availability of precursors, or alternative pathways. Incorporation of other PhL components by the malaria parasite is not as well studied as PC. Exogenous serine, inositol and ethanolamine are incorporated into the corresponding PhL, PE being converted into PC; inositol is only found in PI. Incorporation of ethanolamine >> serine > choline ~ inositol, compared to uninfected erythrocytes (38). The much higher use of ethanolamine is probably due to its role as a known source of PC by transmethylation. Serine shows a high incorporation into PE through decarboxylation of PS, and then subsequent conversion into PC; conversion of PE into PS, performed by polar head group exchange in other organisms, probably does not occur in Plasmodium (2). Both P. falciparum and P. knowlesi synthesize PI from CDP-diacylglycerol and inositol via a PI synthase. The metabolism of PI also includes PI-bisphosphate and the secondary messengers diacylglycerol (DAG) and inositol-1,4,5-triphosphate, as part of an operational polyphosphoinositide cycle (2, 39). A sphingomyelin synthase is present in P. falciparum, and appears developmentally regulated (40,41). Localization to the perinuclear region is unusual, since it is a cis-Golgi resident activity in other cell types. The parasite is able, therefore, to acquire host PhL, and their components, for synthesis of most major PhL types. P. falciparum cultivated in vitro incorporates free fatty acids (FFA) into the major PhL species and other complex lipids (42,43). Infected erythrocytes contain significantly higher acyl-CoA synthetase activity than normal erythrocytes (44). Although Plasmodium fatty acid transfer proteins have not been described, the surface of P. falciparum merozoites shows a 75 kDa heat shock protein cognate, which are known to bind fatty acids (2). The malaria parasite, therefore, is replete with pathways to complete de novo synthesis of phospholipids, as long as a continuing supply of fatty acids, DAG and PhL components is available from host lipid. In P. falciparum and P. knowlesi synthesis of glycolipids increases with intracellular development of the parasite. There is a stage-specificity to some glycolipid classes and differences among P. falciparum strains (45, 46). Distinct differences and changes in the complex lipid composition and their acylation patterns are found among isolated parasites and in the plasma membranes of infected and uninfected erythrocytes. This has been determined for P. chabaudi in the mouse (47), in vitro propagated P. falciparum (48,49) and P. knowlesi in monkeys (50,51).

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Isolated parasites possessed variably increased levels of PC and PI, and greatly decreased SM and PS when compared with uninfected erythrocyte membranes. However, whereas PE levels in isolated parasites were essentially unchanged in P. falciparum and P. chabaudi, P. knowlesi showed significantly higher PE levels than uninfected erythrocytes, confirming earlier findings (52). Total lipid fatty acid composition was dissimilar among P. falciparum, P. chabaudi and P. knowlesi (47, 48,53), especially the major fatty acids (C16:0, C18:0, C~8:1,C~8:2C20:4). Although the unsaturation index of P. knowlesi- and P. chabaudi-infected erythrocytes appeared constant, P. falciparum showed a significant decrease. P. falciparum and P. knowlesi showed a significant decrease in C2o.4. The fatty acid composition of individual P. falciparum phospholipid classes was also examined, and those from parasite and infected erythrocyte membranes were very similar but different from normal erythrocytes. Most striking was a significant increase in C~6:0and C18:2 and a reduction in C20:4, in the PE fraction, particularly of isolated parasites. PE showed the greatest decrease in unsaturation index of any PhL species. Therefore, the FA composition of individual lipid classes, as well as the gross PhL composition of the malaria parasite is substantially different from that of the normal erythrocyte. In addition, the host cell membrane is changed in P. falciparum, which in some cases extends to the PhL molecular species composition. The fatty acid molecular species composition of some PhL from membranes of P. chabaudi- and P. knowlesi-infected erythrocytes has been examined (26, 54). Erythrocyte membranes from P. chabaudi-infected cells were similar in PC molecular species composition to both cohort unparasitized erythrocytes, and erythrocytes from uninfected animals. All changes in PC molecular species composition in infected erythrocytes could be attributed to the parasite. The parasite showed increases in most species containing C20.4, and decreases in those showing only saturated FA. Therefore, at least in P. chabaudi, parasite PC is much less saturated than the host cell membrane. No changes in plasma PC molecular species composition were seen in infected animals. In contrast, P. knowlesi showed a major increase in one PC species, C16:o/18:2, and the host cell membranes from infected erythrocytes displayed alterations in molecular species composition similar to those of the isolated parasite (26). The erythrocyte-bound malaria parasite acquires host lipids by diverse methods and transports to the area of celluar growth. There they are remodeled significantly to meet the parasite's needs, including phospholipid head group alteration, modification of the fatty acid molecular species composition of certain phospholipids, and transformation of the erythrocyte membrane lipid composition in both infected and uninfected cells.

3. EXTRACELLULAR PARASITES As the only unicellular parasite to live free in the host's bloodstream the African, or salivarian, trypanosomes are unique. Unfortunately, there is little information on African trypanosome lipids and lipid metabolism. The life cycle of the African trypanosome involves bloodstream forms in the mammalian host and various insect forms in the tsetse fly vector. Of the insect forms, only the in vitro propagated procyclic form, equivalent to those in the tsetse fly midgut, has been examined. Early studies used complex culture medium that contained significant exogenous lipid. Later studies used

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more defined media, which usually included fetal bovine serum. Differences in bloodstream and procyclic form lipid composition can be due to differences in individual isolates and, in the case of procyclic forms, the medium used for cultivation. Dixon and Williamson (55) analysed the lipid composition of T. brucei rhodesiense bloodstream and procyclic forms. Although sterol esters (SE), FFA and sterols were about equal between the two stages, procyclic forms contained much more triacylglycerol (TAG) than bloodstream forms. Both life cycle stages contained similar amounts of PC, but procyclic forms contained more PE and far less SM than bloodstream forms. When the fatty acid (FA) content of bloodstream and procyclic forms was compared with that of host plasma and culture medium, respectively, significant differences were seen. Bloodstream forms showed less C16:0 and C18:1 than their environment, but greatly increased polyunsaturated fatty acids, namely C18:2 and C22.6. Procyclic forms showed much lower C16:0 and higher C18:0 levels than unused medium whereas C18.1 was decreased and C18.2 was much higher than that supplied. Curiously, these forms also possessed C22:5, which was not present in preculture medium. Therefore, both bloodstream forms and in vitro propagated procyclic forms significantly increase their overall FA unsaturation over that available, indicating an ability to desaturate acyl chains. Significant chain elongation was also apparent. The complex lipid composition of T. b. brucei bloodstream forms has been examined (56). Phospholipids showed a distribution of PC>>PE>>SM > PI ,~ PS, whereas in neutral lipid, Cho>>FFA > TAG ~ SE. In general this PhL distribution, except for SM, has been confirmed (57, 58). The total lipid FA composition of the Trypanozoon subspecies T. b. brucei, T. b. 9ambiense and T. b. rhodesiense are similar and all species showed a high degree of FA unsaturation (59). Dixon et al. (60) assessed the incorporation of radiolabeled glucose, glycerol and acetate into the complex lipids and FA of T. b. rhodesiense bloodstream and procyclic forms. Procyclics showed a higher incorporation of glucose into total lipid than bloodstream forms and were able to incorporate acetate into FA, with incorporation into saturates >>trienes >>monoenes/dienes. Incorporation into different chain length FA showed C18 > C22 ~ C14 > C16 > C20. Bloodstream forms were able to accomplish significant FA chain elongation and desaturation. Both bloodstream and procylic forms were able to oxidize FA, with procyclic forms showing higher activity. Procyclics generally showed a greater capacity than bloodstream forms at incorporating FA into complex lipids and, in both forms, incorporation into PhL was generally greater than into neutral lipid. Bloodstream forms consistently incorporated more total FA into phospholipids than procyclic forms, whereas the latter showed higher levels in neutral lipid. In general, procyclic forms incorporated FA into PC>> PI > PE, whereas bloodstream forms reversed the order of PI and PE. These studies pointed out at an early date that the FA and complex lipid metabolism of the two life-cycle stages were significantly different. Venkatesan and Ormerod (61) examined lipid composition differences between two different bloodstream form stages of T. b. rhodesiense, the long slender form that is actively dividing, and the 'stumpy' form, a non-dividing form that differentiates from long slender forms. Although similar in the percentage of some lipid classes, the stumpy forms showed elevated total lipid, total neutral lipid, sterol ester, and PhL; PI was decreased, compared to slender forms. Stumpy forms also contained plasmalogens (alk-l-enyl ether linkage of FA) in the phospholipid fraction. The total polar lipid FA

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of both forms were similar. The polar lipid of both forms, when compared to host plasma, showed significant decreases in C16:0and 20-carbon polyenoic FA, whereas C18:2 and 22-carbon polyenoic FA were greatly increased. Stumpy forms showed cholesterol ester with C~6:0,C~8:0,C~8:~,C18:2, C22:4and C22:5increased over that of the host; 20-carbon polyenoics and C22:6 were decreased. The proportion of most FA could be modulated by feeding the host (rat) a fat-deficient diet. This supported earlier studies which suggested that the bloodstream form trypanosome obtains much of its lipid from the host and that it is significantly remodeled. These observations are interesting because the stumpy forms of pleomorphic salivarian trypanosomes are thought to be 'primed' for differentiation into procyclic forms after the tsetse fly takes a bloodmeal. When tsetse flies were fed on T. congolense procyclic forms suspended in a medium containing normal or delipidated fetal bovine serum, comparable midgut infections developed (62). However, flies fed on lipid-free medium showed significantly lower infection levels in the labrum and hypopharynx (epimastigotes and metacyclics). Therefore, lipid is necessary to achieve successful metacyclogenesis and continuation of the life cycle. The fatty acids of the bloodstream forms of salivarian trypanosomes appear, on the most part, to be qualitatively similar to their host, although the quantitative FA composition of some lipid classes is much different. The African trypanosome seems to possess the ability to rearrange the FA composition of individual complex lipid classes and, through chain elongation and desaturation to modify host-acquired FA. Procyclic forms do not synthesize cholesterol de novo but can incorporate this sterol if supplied exogenously; de novo synthesis of ergosterol does occur. Bloodstream forms are able to acquire and incorporate the major mammalian bloodstream sterol into their membranes although they possess no ability to synthesize sterol de novo (63). The method of acquiring host cholesterol is of major significance. Both life cycle stages of T. b. brucei showed a significant and biphasic uptake of FA from the external medium, which was promoted by the presence of bovine serum albumin. The rate of FA uptake by culture forms was almost 16-fold greater than bloodstream forms of the same clone. The reversibility of the process was noted in the presence or absence of bovine serum albumin, suggesting a direct interaction of the parasite with bovine serum albumin and FA-albumin complexes. The finding that procyclic trypanosomes take up significantly more exogenous FFA than bloodstream forms is even more curious when one examines the literature on de novo FA biosynthesis by procyclic forms. T. brucei procyclic forms utilized large amounts of threonine from a synthetic culture medium; the products of this process were glycine and acetate, which were excreted in nearly equimolar amounts. It was postulated that threonine was the preferred substrate for acetate production and, although much acetate was excreted, it could be used for FA synthesis. The enzymes responsible were on NAD +-dependent L-threonine dehydrogenase and aminoacetone synthase (acetylCoA:glycine C-acetyltransferase). Subsequently it was shown (64, 65) that threonine was the preferred substrate for acetate production even in the presence of excess acetate or glucose. Incorporation into lipid was over the in vitro growth cycle, and threonine contributed the majority of carbon atoms to palmitic and stearic acids. A known inhibitor of L-threonine dehydrogenase decreased the production of acetate and glycine from L-threonine, decreased incorporation of label from threonine into lipid and was trypanocidal. Additional investigation into threonine catabolism in the African

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trypanosomes showed that bloodstream forms also possessed this pathway (66). Therefore, bloodstream forms could obtain acetate units from threonine which explains the fact that bloodstream forms are able to elongate fatty acids chains (67). The two enzymes must be closely linked in vivo and are found in the mitochondrial matrix (68). There are few further assessments of the biosynthetic capabilities of procyclic forms. Opperdoes (69) performed a detailed study on the presence and intracellular localization of alkoxyphospholipid biosynthesis enzymes in T. brucei procyclic forms. The complete pathway was reported to be localized in the microbody or 'glycosome' fraction, as were all other enzymes essential for de novo synthesis of this lipid class from glycerol and acyl-CoA (see Chapter 2). These pathways also exist in bloodstream forms. As obligate blood parasites with limited de novo synthetic capabilities, both the bloodstream form of African trypanosomes and the intracellular malaria parasite must acquire and remodel host complex lipids. Plasmodium sp. possesses enzymic activities, particularly those involving PC and PE, that allow them to synthesize complex phospholipids. The African trypanosome must also obtain and synthesize complex lipids but has evolved a different strategy, which has been admirably reviewed by Mellors and Samad (70). Early studies were concerned with the anemia associated with trypanosomiasis, perhaps caused by free fatty acids and lysophospholipids, as the products of phospholipases. Phospholipase A 1 is found in plasma and tissue fluids of infected animals and cleaves acyl chains from both exogenous lysophospholipids and PC, yielding both free fatty acids and 2-1ysophosphatidylcholine. The latter lipid is then rearranged to a 1-acyl derivative, and is again acted upon by phospholipase A 1. Procyclic forms show much less activity than bloodstream forms, where it is associated with the plasma membrane. Samad et al. (71) identified considerable acyl-CoA-dependent acyltransferase and acyl-CoA hydrolase activities in bloodstream forms of T. brucei. The former enzyme can acylate exogenous lysophospholipid with the specificity: lysoPC > lysoPI > lysoPE > lysoPA > lysoPS. The enzyme is membrane-bound and shows a preference for CoAacyl thioesters different from that of mammalian acyl transferases which are frequently specific for arachidonate (C20:4). The acyl-CoA hydrolase activity is most likely cytoplasmic in origin and does not appear to be involved in PhL synthesis. Besides acyltransferase and phospholipase A1, an acyl-CoA ligase has been described; all three enzymes are localized in the plasma membrane. Procyclic forms possess little activity of any of the three enzymes which is concordant with the greater biosynthetic power of the insect form (72). Interconversion of diacyl phospholipids is suggested by the above (70, 71), as is PC generated by transmethylation of PE, and PE by decarboxylation of PS, although the amounts produced are comparable to that from transacylation. From these studies it emerges that the lipid biosynthetic pathways of the African trypanosomes are complex, differing both from the host and the malaria parasite, and rely heavily on an external source of preformed lipid. Host complex lipids bound to serum proteins, most likely lipoproteins and particularly HDL, are a source of lipid for African trypanosomes as they appear to be for Plasmodium. Bloodstream forms of T. b. brucei propagated in vitro under axenic conditions will not continuously divide without exogenously supplied LDL or HDL (73); apolipoproteins do not support growth and anti-apolipoprotein antibodies inhibited growth. LDL- and HDL-bound cholesterol, cholesterol ester, cholesterol oleoyl ether and PC

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were all accumulated, and some metabolized, by the parasite. In most cases uptake was more efficient from HDL. Cholesterol ester was cleaved to free cholesterol and FA by actively dividing, long slender trypomastigotes, although this life-cycle stage possessed only a limited ability to re-esterify cholesterol. This suggests that cholesterol ester serves as a source for bulk sterol, and for acyl chains for the PhL remodeling reactions described above. In contrast, short stumpy forms of the parasite showed efficient esterification of acyl chains to cholesterol, supporting the earlier observations of Venkatesan and Ormerod (61) that stumpy forms contain increased amounts of SE. Vandeweerd and Black (74) also noted that lipoprotein-bound PC was acquired and metabolized by bloodstream forms and the conversion products did not show lysophosphatidylcholine. Therefore, there is firm evidence that the salivarian trypanosomes acquire, and subsequently modify, host-derived lipids, the majority of which are obtained from LDL or HDL. Stage-specific differences in de novo lipid synthesis are well supported. Procyclic forms are able to synthesize, elongate and desaturate FA, and to synthesize sterol (ergosterol). The lack of sterol synthesis by bloodstream forms is firmly established (see also chapter 11).

4. ACKNOWLEDGEMENTS This chapter is dedicated to the late Dr George G. Holz, J r - - m e n t o r , colleague and friend. The author thanks Dr H. J. Vial (Montpellier, France) for prepublication manuscripts, and Drs I. W. Sherman (Irvine, CA, USA) and A. Mellors (Guelph, Ontario, Canada) for comments on this chapter. Mr Castor Wamukoya Kweyu (ILRAD, Nairobi, Kenya) is thanked for help in gathering the materials needed to write this chapter.

5. REFERENCES 1. Simoes, A. P., Roelofsen, B. and Op den Kamp, J. A. F. (1992) Lipid compartmentalization in erythrocytes parasitized by Plasmodium-spp. Parasitol. Today 8: 18-21. 2. Vial, H. J. and Ancelin, M. L. (1992) Malarial lipids. An overview. Subcell. Biochem. 18: 259-306. 3. Smith, J. D. (1993) Phospholipid biosynthesis in protozoa. Prog. Lipid Res. 32: 47-60. 4. Holder, A. A., Blackman, M. J., Borre, M. et al. (1994) Malaria parasites and erythrocyte invasion. Biochem. Soc. Trans. 22: 291-295. 5. Joiner, K. A. (1991) Rhoptry lipids and parasitophorous vacuole formation - - a slippery issue. Parasitol. Today 7: 226-227. 6. Bannister, L. H. and Dluzewski, A. R. (1990) The ultrastructure of red cell invasion in malaria infections: a review. Blood Cells 16: 257-292. 7. Gratzer, W. B. and Dluzewski, A. R. (1993) The red blood cell and malaria parasite invasion. Semin. Hematol. 30: 232-247. 8. Mikkelsen, R. B., Kamber, M., Wadwa, K. S., Lin, P.-S. and Schmidt-Ullrich, R. (1988) The role of lipids in Plasmodium falciparum invasion of erythrocytes: a coordinated biochemical and microscopical analysis. Proc. Natl. Acad. Sci. USA 85: 5956-5960. 9. Dluzewski, A. R., Mitchell, G. H., Fryer, P. R., Gritfiths, S., Wilson, R. J. M. and Gratzer, W. B. (1992) Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells. J. Cell Sci. 102: 527-532.

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10. Sjoberg, K., Hosein, Z., Wahlin, B. et al. (1991) Plasmodium falciparum-an invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen. Exp. Parasitol. 73:317-325. 11. Haldar, K. and Uyetake, L. (1992) The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes. Mol. Biochem. Parasitol. 50:161-177. 12. Ward, G. E., Miller, L. H. and Dvorak, J. A. (1993) The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes. J. Cell Sci. 106: 237-248. 13. Stewart, M. J., Schulman, S. and Vanderberg, J. P. (1986) Rhoptry secretion of membranous whorls by Plasmodium falciparum merozoites. Am. J. Trop. Med. Hyg. 35: 37-44. 14. Sam-Yellowe, T. Y. (1992) Molecular factors responsible for host cell recognition and invasion in Plasmodium falciparum. J. Protozool. 39:181-189. 15. Perkins, M. E. and Ziefer, A. (1994) Preferential binding of Plasmodiumfalciparum SERA and rhoptry proteins to erythrocyte membrane inner leaflet phospholipids. Infect. Immun. 62: 1207-1212. 16. Sam-Yellowe, T. Y., Shio, H. and Perkins, M. E. (1988) Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes. J. Cell Biol. 106: 1507-1513. 17. Bonhomme, A., Pingret, L. and Pinon, J. M. (1992) Review: Toxoplasma gondii cellular invasion. Parassitologia 34: 31-43. 18. Joiner, K. A., Beckers, C. J. M., Bermudes, D., Ossorio, P. N., Schwab, J. C. and Dubremetz, J. F. (1994) Structure and function of the parasitophorous vacuole membrane surrounding Toxoplasma gondii. Ann. N YAcad. Sci. 730: 1-6. 19. Foussard, F., Lerichie, M. A. and Dubremetz, J. F. (1991) Characterization of the lipid content of Toxoplasma gondii rhoptries. Parasitology 102: 367-370. 20. Halonen, S. K. and Weidner, E. (1994) Overcoating of Toxoplasma parasitophorous vacuoles with host cell vimentin type intermediate filaments. J. Euk. Microbiol. 41: 65-71. 21. Saffer, L. D. and Schwartzman, J. D. (1991) A soluble phospholipase of Toxoplasma gondii associated with host cell penetration. J. Protozool. 38: 454-460. 22. Etzion, Z., Murray, M. C. and Perkins, M. E. (1991) Isolation and characterization of rhoptries of Plasmodium falciparum. Mol. Biochem. Parasitol. 47:51-62. 23. Vial, H. J., Ancelin, M.-L., Philippot, J. R. and Thuet, M. J. (1990) Biosynthesis and dynamics of lipids in Plasmodium-infected mature mammalian erythrocytes. Blood Cells 16: 531-555. 24. Grellier, P., Rigomier, D., Clavey, V., Fruchart, J.-C. and Schrevel, J. (1991) Lipid traffic between high density lipoproteins and Plasmodiumfalciparum-infected red blood cells. J. Cell Biol. 112: 267-277. 25. Valentin, A., Rigomier, D., Precigout, E., Carcy, B., Gorenflot, A. and Schrevel, J. (1991) Lipid trafficking between high density lipoproteins and Babesia divergens-infected human erythrocytes. Biol. Cell 73: 63-70. 26. Simoes, A. P., Moll, G. N., Beaumelle, B., Vial, H. J., Roelofsen, B. and Op den Kamp, J. A. F. (1990) Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes. Biochim. Biophys. Acta 1022: 135-145. 27. Pouvelle, B., Spiegel, R., Hsiao, L. et al. (1991) Direct access to serum macromolecules by intraerythrocytic malaria parasites. Nature 353: 73-75. 28. Sherman, I. W. and Zidovetzki, R. (1992) A parasitophorous duct in Plasmodium-infected red blood cells. Parasitol. Today. 8: 2-3. 29. Haldar, K. (1992) Lipid transport in Plasmodium. Infect. Ag. Dis. 1: 254-262. 30. Grellier, P., Rigomier, D. and Schrevel, J. (1990) Induction in vitro de la schizogonie de Plasmodium falciparum par les lipoproteines humaines de haute densite (HDL). C R Acad. Sci. Paris 311 Serie III: 361-367. 31. Ancelin, M. L., Parant, M., Thuet, M. J., Philippot, J. R. and Vial, H. J. (1991) Increased permeability to choline in simian erythrocytes after Plasmodium knowlesi infection. Biochem. J. 273: 701-709. 32. Kirk, K., Wong, H. Y., Elford, B. C., Newbold, C. I. and Ellory, J. C. (1991) Enhanced choline and Rb+transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum. Biochem. J. 278: 521-525.

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33. Kirk, K., Poli de Figueiredo, C. E., Elford, B. C. and Ellory, J. C. (1992) Effect of cell age on erythrocyte choline transport: implications for the increased choline permeability of malariainfected erythrocytes. Biochem. J. 283: 617-619. 34. Ancelin, M. L. and Vial, H. J. (1992) Saturable and non-saturable components of choline transport in Plasmodium-infected mammalian erythrocytes: possible role of experimental conditions. Biochem. J. 283: 619-621. 35. Ancelin, M. L. and Vial, H. J. (1986) Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine. FEBS Lett. 202:217-223. 36. Vial, H. J., Thuet, J. J. and Philippot, J. R. (1984) Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers. Biochim. Biophys. Acta 795: 372-383. 37. Ancelin, M. L. and Vial, H. J. (1989) Regulation of phosphatidylcholine biosynthesis in Plasmodium knowlesi-infected erythrocytes. Biochim. Biophys. Acta 1001: 82-89. 38. Elabbadi, N., Ancelin, M. L. and Vial, H. J. (1992) Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial activity by the semiautomated microdilution technique. Antimicrob. Agents Chemother. 36: 50-55. 39. Elabbadi, N., Acelin, M. L. and Vial, H. J. (1994) Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositide cycle in Plasmodium-infected erythrocytes. Mol. Biochem. Parasitol. 63:179-192. 40. Elmendorf, H. G. and Haldar, K. (1993) Identification and localization of ERD2 in the malaria parasite Plasmodiumfalciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi. EMBO J. 12: 4763-4773. 41. Elmendorf, H. G. and Haldar, K. (1994) Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubulovesicular network in the cytoplasm of mature erythrocytes. J. Cell Biol. 124: 449-462. 42. Vial, H. J., Ancelin, M. L., Thuet, M. J. and Philippot, J. R. (1989) Phospholipid metabolism in Plasmodium-infected erythrocytes: guidelines for further studies using radioactive precursor incorporation. Parasitology 98:351-357. 43. Dieckmann-Schuppert, A., Bender, S., Holder, A. A., Haldar, K. and Schwarz, R. T. (1992) Labeling and initial characterization of polar lipids in cultures of Plasmodium falciparum. Parasitol. Res. 78:416-422. 44. Beaumelle, B. D. and Vial, H. J. (1988) Acyl-CoA synthetase activity in Plasmodium knowlesi infected erythrocytes displays peculiar substrate specificities. Biochim. Biophys. Acta 958: 1-9. 45. Sherwood, J. A. Spitalnik, S. L., Aley, S. B., Quakyi, I. A. and Howard, R. J. (1986) Plasmodiumfalciparum and P. knowlesi: initial identification and characterization of malaria synthesized glycolipids. Exp. Parasitol. 62: 127-141. 46. Sherwood, J. A., Spitalnik, S. L., Suarez, S. C., Marsh, K. and Howard, R. J. (1988) Plasmodiumfalciparum glycolipid synthesis: constant and variant molecules of isolates and of strains with differing knob and cytoadherence phenotype. J. Protozool. 35: 169-172. 47. Wunderlich, F., Fiebig, S., Vial, H. and Kleinig, H. (1991) Distinct lipid composition and host cell plasma membranes from Plasmodium chabaudi-infected erythrocytes. Mol. Biochem. Parasitol. 44: 271-278. 48. Hsiao, L. L., Howard, R. J., Aikawa, M. and Taraschi, T. F. (1991) Modification of host cell membrane lipid composition by the intra-erythrocytic human malaria parasite Plasmodium falciparum. Biochem. J. 274: 121-132. 49. Maguire, P. A. and Sherman, I. W. (1990) Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodiumfalciparum-infected red cells. Mol. Biochem. Parasitol. 38: 105-112. 50. Joshi, P., Dutta, G. P. and Gupta, C. M. (1987) An intracellular simian malarial parasite (Plasmodium knowlesi) induces stage-dependent alterations in membrane phospholipid organization of its erythrocyte. Biochem. J. 246: 103-108. 51. Van der Schaft, P. H., Beaumelle, B., Vial, H., Roelofsen, B., Op den Kamp, J. A. F. and Van Deenen, L. L. M. (1987) Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection. Biochim. Biophys. Acta 901: 1-14. 52. McClean, S., Purdy, W. C., Kabat, A., Sampugna, J. and DeZeeuw, R. (1976) Analysis of the phospholipid composition of Plasmodium knowlesi and rhesus erythrocyte membranes. Anal. Chim. Acta 82: 175-185.

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53. Beaumelle, B. D. and Vial, H. J. (1986) Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi. Biochim. Biophys. Acta 877: 262-270. 54. Simoes, A. P., Fiebig, S., Wunderlich, F., Vial, H., Roelofsen, B. and Op den Kamp, J. A. F. (1993) Plasmodium chabaudi-parasitized erythrocytes: phosphatidylcholine species of parasites and host cell membranes. Mol. Biochem. Parasitol. 57: 345-348. 55. Dixon, H. and Williamson, J. (1970) The lipid composition of blood and culture forms of Trypanosoma lewisi and Trypanosoma rhodesiense compared with that of their environment Comp. Biochem. Physiol. 33:111-128. 56. Carroll, M. and McCrorie, P. (1986) Lipid composition of bloodstream forms of Trypanosoma brucei brucei. Comp. Biochem. Physiol. 83B: 647-651. 57. Doering, T. L., Pessin, M. S., Hoff, E. F., Hart, G. W., Raben, D. M. and Englund, P. T. (1993) Trypanosome metabolism of myristate, the fatty acid required for the variant surface glycoprotein membrane anchor. J. Biol. Chem. 268: 9215-9222. 58. Patnaik, P. K., Field, M. C., Menon, A. K., Cross, G. A. M., Yee, M. C. and Butikofer, P. (1993) Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms. Mol. Biochem. Parasitol. 58: 97-105. 59. Breton, J. C., Jauberteau, M. O., Rigaud, M. et al. (1988) Les acides gras de Trypanosoma brucei brucei. Etude comparative de T. b. rhodesiense et T. b. gambiense. Bull. Soc. Pathol. Exot. Filiales 81: 632-636. 60. Dixon, H., Ginger, C. D. and Williamson, J. (1971) The lipid metabolism of blood and culture forms of Trypanosoma lewisi and Trypanosoma rhodesiense. Comp. Biochem. Physiol. 39B: 247-266. 61. Venkatesan, S. and Ormerod, W. E. (1976) Lipid content of the slender and stumpy forms of Trypanosoma brucei rhodesiense: a comparative study. Comp. Biochem. Physiol. 53B: 481-487. 62. Maudlin, I., Kabayo, J. P., Flood, M. E. T. and Evans, D. A. (1984) Serum factors and the maturation of Trypanosoma congolense infections in Glossina morsitans. Z. Parasitenk. 70: 11-19. 63. Dixon, H., Ginger, C. D. and Williamson, J. (1972) Trypanosome sterols and their metabolic origins. Comp. Biochem. Physiol. 41B: 1-18. 64. Klein, R. A. and Cross, G. A. M. (1976) Threonine as a two-carbon precursor for lipid synthesis in Trypanosoma brucei. Trans. R. Soc. Trop. Med. Hyg. 69: 267. 65. Klein, R. A. and Linstead, D. J. (1976) Threonine as a preferred source of 2-carbon units for lipid synthesis in Trypanosoma brucei. Biochem. Soc. Trans. 4: 48-50. 66. Linstead, D. J., Klein, R. A. and Cross, G. A. M. (1977) Threonine catabolism in Trypanosoma brucei. J. Gen. Microbiol. 101: 243-251. 67. Klein, R. A. and Miller, P. G. G. (1981) Alternate pathways in protozoan energy metabolism. Parasitology 82: 1-30. 68. Opperdoes, F. R., Markos, A. and Steiger, R. F. (1981) Localization of malate dehydrogenase, adenylate kinase and glycolytic enzymes in glycosomes and the threonine pathway in the mitochondrion of cultured procyclic trypomastigotes of Trypanosoma brucei. Mol. Biochem. Parasitol. 4: 291-309. 69. Opperdoes, F. R. (1984) Localization of the initial steps in alkoxyphospholipid biosynthesis in glycosomes (microbodies) of Trypanosoma brucei. FEBS Lett. 169: 35-39. 70. Mellors, A. and Samad, A. (1989) The acquisition of lipids by African trypanosomes. Parasitol. Today 5: 239-244. 71. Samad, A., Licht, B., Stalmach, M. E. and Mellors, A. (1988) Metabolism of phospholipids and lysophospholipids by Trypanosoma brucei. Mol. Biochem. Parasitol. 29: 159-169. 72. Bowes, A. E., Samad, A. H., Jiang, P., Weaver, B. and Mellors, A. (1993) The acquisition of lysophosphatidylcholine by African trypanosomes. J. Biol. Chem. 268: 13885-13892. 73. Black, S. and Vandeweerd, V. (1989) Serum lipoproteins are required for multiplication of Trypanosoma brucei brucei under axenic culture conditions. Mol. Biochem. Parasitol. 37: 65-72. 74. Vandeweerd, V. and Black, S. J. (1989) Serum lipoprotein and Trypanosoma brucei brucei interactions in vitro. Mol. Biochem. Parasitol. 37: 201-212.

9

Antioxidant Mechanisms ROBERTO DOCAMPO Department of Veterinary Pathobiology, University of Illinois, Urbana, III, USA

SUMMARY Because they live in oxygen-poor environments, parasites are particularly sensitive to oxidative stress. Catalase is absent in many. Glutathione peroxidase activities are very low or absent, and in some cases, such as in trypanosomatids, replaced by trypanothione-dependent peroxidase activities. In any case, peroxidase activities are quite low when compared with the glutathione peroxidase activities in mammalian organs. Superoxide dismutases, however, are present in most parasites, and these are features typical of facultative aerobes. Since superoxide dismutase catalyzes the dismutation of superoxide anion to form hydrogen peroxide and there is a deficiency in hydrogen peroxide-metabolizing systems, this explains why superoxide anion is so toxic to parasites. Parasites may tolerate a slow endogenous rate of hydrogen peroxide generation in their natural habitats but they are quite sensitive to an increased steady-state concentration of hydrogen peroxide as a result of drug metabolism or phagocytic cell attack.

1. INTRODUCTION In aerobic organisms, molecular oxygen is a final acceptor of electrons in different electron transport systems, such as those present in mitochondria. Much experimental evidence now supports the thesis that the favorable aspects of aerobic life are also linked to potentially dangerous oxygen-linked processes. Several highly unstable and reactive oxygen-derived free radicals are produced during oxygen metabolism and oxidative processes in living cells; these include the superoxide anion (O2), produced by the univalent reduction of molecular oxygen, and the highly toxic hydroxyl radical Copyright 9 1995 Academic Press Ltd All rights of reproduction in any form reserved

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(OH'), which can be produced from hydrogen peroxide (H202) and the superoxide anion in the presence of transition metal ions. Oxidative damage caused by reactive oxygen species has been called 'oxidative stress'. Biological systems contain powerful enzymatic and non-enzymatic antioxidant mechanisms, and oxidative stress denotes a shift in the pro-oxidant/antioxidant balance in favor of the former (1). Reactive oxygen species are important mediators of several forms of cell damage in parasites and this discussion will focus on the defense mechanisms of parasites against these species.

2. TOXICITY OF OXYGEN IN PARASITES A number of parasites live in oxygen-poor environments and are particularly sensitive to oxidative stress. Obligate anaerobes, which lack defenses against reactive oxygen species, are killed readily by oxygen at tensions higher than those in their natural habitats. Only two groups of protozoa, rumen ciliates and termite flagellates (2), belong to this category. It has been shown that rumen protozoa are able to respire under low 02 tensions (1-2 kPa (kiloPascals) of 02), where they exhibit a relatively high apparent K m for 02, whereas at 02 tensions above 2 kPa the respiration decreases rapidly, indicating 02 toxicity. Since respiration at low 02 tensions is stable for several hours, it may be of importance to the organism at the low 02 concentrations of rumen fluid (3). Other protozoa are less sensitive to oxygen damage and consume oxygen when available; they can be cultivated for indefinite periods under completely or nearly anaerobic conditions. Examples are Entamoeba spp. and Trichomonas spp. However, growth of Trichomonas vaginalis is stimulated by the presence of low oxygen tensions (1-3/~M) when compared to growth under anaerobiosis; this characteristic defines this organism as a microaerophile (4). Trypanosomatids are also able to survive under anaerobic conditions but, in contrast to the aforementioned forms, they cannot multiply in anaerobiosis (2). All parasitic helminths studied thus far are facultative aerobes, showing various degrees of tolerance to a lack of oxygen. Because Fasciola hepatica and large intestinal helminths (Ascaris, Monieza, and others) live in oxygen-poor environments, their oxygen consumption is strictly dependent on the oxygen tensions and on their K m for 02. Most authors assume that they lead a predominantly anaerobic life in vivo (2) (Chapter 4). For experimental purposes, parasites are frequently tested in media with greater oxygen tensions than their oxygen-poor natural environments. This sudden increase in the partial pressure of oxygen may result in metabolic changes analogous to those that occur when aerobic organisms are placed in hyperbaric oxygen. In fact, any gas mixture with Po 2 greater than that in which organisms are adapted may be considered hyperbaric oxygenation (5). The high sensitivity of parasites to reagent H202, to different 0 2 - (such as autoxidation of thiols, xanthine-xanthine oxidase, dihydro-orotate-dihydro-orotate oxidase) and H202-generating systems (such as glucose-glucose oxidase), and organic peroxides (such as tert-butyl hydroperoxide) is well established (5). In addition, their sensitivity to both drug-generated and phagocyte-derived oxygen reactive species is also well documented (5, 6). It seems possible that 0 2 and H202 are damaging not by virtue

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of direct attack upon parasite components, but rather because they can give rise to the extremely reactive hydroxyl radical (OH-) in the presence of transition metal ions (Fenton reaction): O2 + M "+a ~ M" +

0 2

M" + H 2 0 2 ~ M "+ 1 + O H - + OH" 0 2 + H 2 0 2 ~ 0 2 + O H - + OH"

Recently, a novel mechanism for hydroxyl radical production, which is not dependent on the presence of transition metal ions, has been proposed (7). This involves the production of peroxynitrite ( O N O O - ) arising from the reaction of nitric oxide (NO') with superoxide (O2), as shown in the following reactions: NO. + O2 ~ O N O O O N O O - + H + --, O N O O H O N O O H ~ O H . + N O 2" The combination of NO. with superoxide competes with the dismutation of superoxide: 2H + + O2 + O~ ~ H 2 0 2 +

0 2

Peroxynitrite has a pKa of 7.5 and will be therefore substantially protonated at physiological pH, to produce peroxynitrous acid (ONOOH). One route for the decomposition of this molecule is thought to be homolytic cleavage to produce hydroxyl radical (OH.) and nitrogen dioxide ( N O z"). Currently, there has been much interest in the biologic role of N O - a s a widespread transduction mechanism leading to a variety of functions in different cells. It is now generally accepted that NO. is the endothelium-derived relaxing factor and that it also participates in the regulation of nervous and immune systems (8, 9). Activated macrophages form NO2 and NO3 from the terminal guanidino nitrogen atom(s) of L-arginine by a process now known to proceed via the formation of NO'. It has been postulated that the macrophage leishmanicidal activity induced by interferon-7 (IFN-7) and lipopolysaccharide (LPS) (10) or by calcium ionophore A23187 in the presence of LPS (11) is mediated by the L-arginine:NO" pathway and it has been demonstrated that the leishmanial parasite can be killed by NO. in cell-free suspensions (10). The trypanocidal activity of IFN-7-treated macrophages against T. cruzi also involves an L-arginine-dependent, nitric oxide-mediated mechanism (12). A nitric oxide-mediated cytostatic activity on T. brucei has also been reported (13) and peroxynitrite has been shown to kill T. cruzi epimastigotes in a dose-dependent manner (14). The precise mechanism of the microbicidal effect of NO. is at present unclear. However, in addition to its capacity to generate hydroxyl radical, it has been shown that exposure of F e - S groups to NO. results in iron-nitrosyl complex formation, which causes inactivation of several enzymes (15).

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3. REACTIVE OXYGEN SPECIES IN SPECIFIC PARASITES Reactive oxygen species are known to play important roles in many biochemical reactions that maintain normal cell functions. These species are produced as normal intermediates in mitochondrial and microsomal electron-transport systems, and some enzymes that produce substantial amounts of 0 2 or H202 have been identified in several parasites (5). Tritrichomonas foetus aerobically excretes H202 (16). In contrast, respiring Trypanosoma cruzi does not release either 0 2 or H202 to the suspending medium, although T. cruzi homogenates supplemented with reduced nicotinamide adenine dinucleotides are effective sources of 0 2 and H202 (5). The mitochondrial, microsomal, and supernatant fractions of T. cruzi also generate O~ and H202 in the presence of NADH or NADPH. Mitochondrial preparations of the T. brucei bloodstream form produce 0.2-0.7 nmol H202 per mg protein/min, which corresponds to 1-3% of the oxygen consumed. Although initial reports set the internal H202 concentration of T. brucei bloodstream forms to a high level (70 pM), further work reported values below the limit of detection. This implies an H202-metabolizing activity in these cells (17). H202 formation by mitochondrial preparations of the protozoan C. fasciculata is qualitatively the same as that by mitochondria from vertebrate tissues (18). In contrast, it has been postulated that in the mitochondria of T. brucei procyclic trypomastigotes (19) and T. cruzi epimastigotes (20) an NADH-fumarate reductase could use 02 as electron acceptor instead of fumarate by converting it into O2 which dismutates to generate HaO 2. A succinate-dependent site for H202 production, which may be the semiquinone of coenzyme Q9, as in mammalian mitochondria, has also been postulated to exist in the mitochondria of T. cruzi epimastigotes (20). Although no direct demonstration of 0 2 or HaO 2 formation by Plasmodium has been reported, a Plasmodium-induced erythrocyte oxidant sensitivity has been postulated (5). During its development inside the erythrocyte the parasite ingests and degrades hemoglobin to obtain essential amino acids. Hemoglobin breakdown in the food vacuole releases heme which is then incorporated into an insoluble material called hemozoin or malaria pigment. It has been postulated that release of low levels of heme or other iron-containing products within the infected erythrocytes could lead to the formation of reactive oxygen species (21,22). In contrast to the mammalian succinate oxidase, this mitochondrial complex in large helminths such as F. hepatica, Ascaris lumbricoides and Monieza expansa, forms H202 from oxygen instead of water and is insensitive to cyanide, azide and antimycin. In addition, there is evidence that the mitochondria of these large helminths contain a functional branched respiratory chain system with two terminal oxidases: cytochrome o and a 3. The major pathway with cytochrome o as its terminal oxidase is apparently linked with HaO2 formation (23). The utilization of NAD(P)H and succinate by Hymenolepis diminuta mitochondrial membranes also results in H202 formation, although the presence of a branched respiratory chain in this cestode has not been established (24). Fumarate reductase has also been implicated as participating in the formation of HaO 2 by M. expansa by a mechanism similar to that described in trypanosomatids (25). Non-enzymatic sources of 0 2 and H20 z, such as the autoxidation of ferredoxins (present in anaerobic parasites such as Trichomonas vaginalis), tetrahydropteridines,

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thiols or other soluble parasite constituents, are of unknown importance under physiologic conditions.

4. ENZYMATIC DEFENSES OF PARASITES AGAINST REACTIVE OXYGEN SPECIES 4.1. Catalase Catalase exerts a dual function: (1) decomposition of H202 to give H20 and 0 2 (catalatic activity) and (2) oxidation of electron donors, e.g., methanol, ethanol, formic acid, phenols, with the consumption of 1 mol of peroxide (peroxidatic activity): 2H202 ~ 2 H 2 0 -+- 0 2

(catalatic activity)

ROOH + A H 2 ~ H 2 0 + ROH + A

(peroxidatic activity)

Catalase is present in the cytoplasm of T. foetus (26) but absent from other Trichomonas species, Giardia lamblia (27) and E. histolytica (5). Although catalase is absent in most pathogenic trypanosomatids such as T. brucei, T. cruzi, and most Leishmania spp., it is present in other mammalian trypanosomes as well as avian, amphibian and insect trypanosomes (2). Although a high content of catalase has been reported in the intracellular (amastigote) forms of L. donovani (28), it is difficult to exclude the possibility that it may be a host contaminant because amastigotes were obtained from hamster spleen homogenates and it is known that catalase can be easily adsorbed to cell membranes. Catalase is localized in most cells in subcellular organelles such as the peroxisomes (microbodies) of liver and kidney or in much smaller aggregates such as the microperoxisomes found in a variety of other cells. There are few reports of peroxisomes or catalase-containing microbodies in parasites. Catalase activity has been detected and localized by biochemical techniques in the microbodies (glycosomes) of some trypanosomatids such as Crithidia luciliae (29), and Phytomonas sp. (30). Although a 3,3'-diamionobenzidine reaction (a cytochemical technique widely used to demonstrate peroxidase activity in microbodies) has been reported in the denser area (core) of hydrogenosomes (31) and in T. cruzi microbodies (32), biochemical determinations were negative and the presence of other peroxidase activities could have been responsible for those positive reactions. The 3,3'-diaminobenzidine-positive microbodies of typical structure are present in lower trypanosomatids. For example, Crithidia fasciculata possesses a high activity of catalase and their catalase-containing microbodies occupy about 2.5% of the total cell volume (5, 33). A low catalase activity has been found in sporulated oocysts and in sporozoites of Eimeria tenella (34) but its location was not determined. Only low levels of catalase were found in P. berghei, probably as a result of contamination of the preparations with host cell material (35). Toxoplasma gondii tachyzoites have an abundant catalase activity (36). Catalase activity is low in trematodes, cestodes and nematodes (2), although it has been detected in significant amounts in the nematodes Ancylostoma ceylanicum and

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Nippostrongylus brasiliensis (37). It is undetectable in newborn larvae, adult worms, and muscle larvae of Trichinella spiralis (38) as well as in F. hepatica and H. diminuta (39). In contrast Helioomosomoides polygyrus contains detectable levels of the enzyme (40). Surprisingly, a substantial catalase activity has been found in isolated A. suum mitochondria (41). 4.2. Glutathione Peroxidase and Other Peroxidase Activities Glutathione peroxidase catalyzes the reaction of hydroperoxides (ROOH) with reduced glutathione (GSH) to form oxidized glutathione disulfide (GSSG) and the reduction product of the hydroperoxides (ROH): ROOH + 2GSH ~ ROH + GSSG + H 2 0 Glutathione peroxidase is specific for its hydrogen donor, reduced glutathione, and non-specific for the hydroperoxide, which may be H202 or organic hydroperoxides (42). Since many of the radical or non-radical reactions in cells may lead to thiol oxidation to the disulfide, i.e., the oxidation of glutathione to form GSSG, the regenerative reaction of rereduction to GSH catalyzed by GSSG reductase can become pivotal in antioxidant defense (1): GSSG + N A D P H + H + ~ 2GSH + NADP + No glutathione peroxidase activity has been detected in E. histolytica and G. lamblia (6). In this regard, it has been pointed out that E. histolytica (43), G. duodenalis and T. foetus (44) lack glutathione metabolism, and that cysteine is the main thiol component present in these parasites. T. vaginalis also lacks detectable GSSG reductase activity (45). Other thiol-dependent peroxidase and reductase activities have been suggested to be present in G. intestinalis but the nature of the thiol cofactors is not known (46). Trypanosomatids do not have glutathione peroxidase. Some of them have a peroxidase activity dependent on a glutathione-spermidine cofactor termed trypanothione (T(SH)2) (47) (see Chapter 7). Oxidation of trypanothione leads to the formation of trypanothione disulfide (T(S)2). A trypanothione peroxidase activity using either H202 or organic hydroperoxides has been measured in crude extracts from C. fasciculata and T. brucei but the enzyme is apparently very labile and has not yet been purified (48). The details of the reaction mechanisms are not clear. However, a system analogous to the host glutathione reductase/glutathione peroxidase system utilizing trypanothione has been proposed (48). The following reaction scheme has been proposed for the metabolism of hydroperoxides: ROOH

~/T(SH)2 ~ ,,

t. p e r o x i d a s e ~ _ ROH + H 2 0

I"

NADP + reductase

'~T(S)2/\

NADPH + H +

Recently, Carnieri et al. (49) reported the absence of a trypanothione peroxidase activity in T. cruzi and suggested that oxidation of endogenous reduced thiols accounted for the rates of H202 metabolism detected in intact cells. In contrast,

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trypanothione reductase has been purified to homogeneity from C. fasciculata (50), and T. cruzi (51), and X-ray structure studies have been performed with the enzymes from C. fasciculata (52-54) and T. cruzi (55-57). The enzyme, which has been found located only in the cytosol of T. brucei (58), catalyzes the NADPH-dependent reduction of trypanothione, but not glutathione. Ultimately, dihydrotrypanothione is capable of undergoing a rapid non-enzymatic disulfide exchange reaction with intracellular disulfides (RSSR), among them oxidized glutathione and cysteine (59). This reaction is summarized as: T(SH)2 + RSSR ~ T(S)2 + 2RSH The cloned trypanothione reductase genes from T. congolense (60) and T. cruzi (61) have been expressed in E. coli, and the enzymes overproduced and purified. The trypanothione reductase genes from Crithidiafasciculata (62, 63) and T. brucei (63) have also been cloned and sequenced. Site-directed mutagenesis of either the E. coli glutathione reductase (64) or the T. congolense trypanothione reductase expressed in E. coli (60) has been used to evaluate the role of different residues in the mutually exclusive specificities of trypanothione reductase and glutathione reductase. In the first report (65) on the presence of the thiol-containing cofactor for GSH reduction in trypanosomes that was later identified as trypanothione (47), the authors reported that the addition of L. mexicana ultrafiltrates was able to restore the T. brucei glutathione (trypanothione) reductase activity, thus indicating the presence of trypanothione or a trypanothione-like compound in the leishmanias. An H2 Oz-dependent NADPH oxidation in the presence of trypanothione in ultrafiltrates of L. mexicana amazonensis promastigotes was reported later (66). More recently the gene for the L. donovani trypanothione reductase has been cloned and overexpressed in either L. donovani or T. cruzi (67). Overexpression of trypanothione reductase did not protect either L. donovani promastigotes or T. cruzi epimastigotes against oxidative stress caused by direct exposure of the cells to reagent H202 or t o H2Oz-generating drugs. It is noteworthy that cells overexpressing this enzyme did not show any difference in the amount of reduced thiols as compared to controls and in the metabolism of exogenously added HzO 2 (67) thus confirming previous results (49) indicating that HzO 2 decomposition in T. cruzi is due to non-enzymatic reactions of endogenous reduced thiols with peroxides, and that a true trypanothione peroxidase is absent in these cells. It is difficult to evaluate other reports of the presence of glutathione peroxidase in Leishmania spp., such as one describing the presence of the enzyme in L. tropica promastigotes (67). A high content of glutathione peroxidase has also been reported in L. donovani intracellular forms (28), but it is difficult to rule out a contamination from the host for the same reasons indicated for the high catalase activity detected in these parasites. Other protozoa do not contain trypanothione (65). A glutathione peroxidase activity has been reported in T. gondii tachyzoites (36). The glutathione metabolism of P. falciparum, P. vinckei and P. berghei has been studied. Glutathione peroxidase and glutathione reductase are present in the parasites as is a selenium-independent glutathione peroxidase (68). However, as occurs with the cyanide-sensitive superoxide dismutase present in malaria parasites, the possibility that the parasites store-up host glutathione reductase in the course of intraerythrocyte maturation could not be ruled out (69).

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F. hepatica lacks glutathione peroxidase (39). Glutathione peroxidase activity in Schistosoma mansoni increases significantly as worms mature in their host and is positively correlated to the resistance to antioxidants (70). A glutathione peroxidase gene from S. mansoni has been cloned and sequenced (70). Newborn larvae of T. spiralis lack detectable glutathione peroxidase, whereas, adult worms and muscle larvae contain a high activity of this enzyme (38). In addition, Ancylostoma ceylanicum and Nippostrongylus brasiliensis contain significant glutathione peroxidase activities (37). Other peroxidases less studied are an ascorbate peroxidase present in T. cruzi, cytochrome c peroxidases detected in mitochondrial preparations of C. fasciculata and in A. lumbricoides and F. hepatica, as well as peroxidases demonstrated in F. hepatica, H. diminuta, M. expansa, the hemocele fluid of A. lumbricoides, and the miracidium of S. mansoni (5). Their role in H202 detoxification is unknown.

4.3. Superoxide Dismutases Superoxide dismutase (SOD) is a widely distributed enzyme that exists in a variety of forms. The copper-zinc enzyme (Cu, ZnSOD) is primarily located in the cytosol of eukaryotic cells. Mitochondria contain, in the matrix space, a distinctive cyanideinsensitive manganese-containing enzyme (MnSOD) similar to that found in prokaryotes. In addition, a ferrienzyme (FeSOD) has been identified in bacteria that is also insensitive to cyanide. Amino acid sequence homologies indicate two families of superoxide dismutases. One of these is composed of the Cu, ZnSODs and the other of MnSODs and FeSODs. All these superoxide dismutases catalyze the same reaction (2H + + O2 + O2 ~ H202 + 02) and with comparable efficiency. Surprisingly, it has been reported that G. intestinalis lacks SOD activity (46). If these results are confirmed, it will be a notable exception among eukaryotic organisms. In general, protozoa, as eukaryotic algae, lack the Cu, Zn enzyme. FeSODs have been found in cell lysates of E. dispar and E. histolytica and their genes have been cloned (71). A comparison of the two sequences revealed 5% nucleotide difference resulting in a single amino acid substitution. The primary structure of the enzymes showed the characteristics of FeSODs with approximately 55% similarity with other FeSODs sequences. The enzyme is encoded by single copy genes in both E. dispar and E. histolytica, and no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA (71). SOD activity is present in the facultative anaerobic flagellate T. foetus (72). About five-sixths of the activity of T. foetus is in the non-sedimentable portion of the cytoplasm. The rest is connected with the hydrogenosome. Dialyzed extracts of T. foetus have SOD at substantially higher levels than in other eukaryotic organisms (73). The activity is inhibited by H 2 0 2 and azide but not by cyanide, suggesting that it is FeSOD(s). Three isozymes were seen by isoelectric focusing which appeared to be sensitive to inhibition by HzO 2 (73). A cyanide-sensitive SOD activity has been reported in T. cruzi (74). Cyanideinsensitive SODs are also present in trypanosomatids, such as C. fasciculata (75) and Trypanosoma brucei (76). The bimodal distribution of superoxide dismutase in T. brucei is similar to that described for other eukaryotes. Both SOD activities in T. brucei are insensitive to 1 mM cyanide (76). The SOD activities of T. brucei, L. tropica and T. cruzi are also cyanide-insensitive but peroxide- and azide-sensitive (77). SOD of C. fasciculata is located in the cytosol and exists in three forms, which may represent three

9. ANTIOXIDANT MECHANISMS

15 5

distinct isozymes. Comparisons of the amino acid content of this SOD with those of SODs from other sources suggests that the crithidial enzyme is closely related to bacterial FeSOD and only distantly related to human MnSOD and Cu, ZnSOD and to the FeSOD of the alga Euglena 9racilis. SOD activity has also been detected in L. donovani, although its nature has not been determined (5). P. berohei isolated from mouse red blood cells also contains a cyanide-sensitive SOD activity. Plasmodial and mouse enzymes are indistinguishable electrophoretically. These results suggest that the malarial cyanide-sensitive SOD may be entirely of host origin and stored by the parasite. Accordingly, plasmodium isolated from mouse red blood cells contain mouse cyanide-sensitive SOD, whereas rat-derived parasites contain the rat enzyme (78). Another, peroxide-sensitive, apparently manganese-containing SOD, has been described in P. falciparum (79). Two Babesia species (B. hylomysci and B. divergens) have been reported to contain an endogenous superoxide dismutase that is cyanide-insensitive and inhibited by HzO 2 indicating that iron is the cofactor metal (80). A similar situation has been found in T. 9ondii where the enzyme(s) are inhibited by azide and peroxide but not by cyanide (81). Unsporulated oocysts of E. tenella have high SOD activity and contain several electrophoretically distinct forms of the enzyme, including two forms of Cu, ZnSOD, two forms of FeSOD and two forms of MnSOD. The SOD activity decreases during sporulation and oocysts sporulated for 48 h have low levels of SOD and contain only one form of the enzyme (MnSOD) (34). S. mansoni possess a Cu, ZnSOD (82). This superoxide dismutase may be secreted since the product of in vitro translation of its mRNA is immunoprecipitable with sera pooled from patients with chronic schistosomiasis. Another Cu, ZnSOD, with homology to mammalian cytosolic forms of the enzyme has been recently cloned from S. mansoni (83). Extracts of F. hepatica show appreciable SOD activity (39). SOD activities are also present in H. diminuta extracts and in the A. lumbricoides (5) but they have not been characterized. A cyanide-sensitive SOD has been purified from metacestodes of Taenia taeniformis (84). SOD activity has also been detected in newborn larvae, adult worms, and muscle larvae of T. spiralis, the activity of newborn larvae being significantly lower than that of the other stages (38). A Cu, ZnSOD has been isolated and purified from muscle larvae of T. spiralis that is apparently secreted extracellularly (85). A Cu, ZnSOD from Onchocerca volvulus has been cloned (86). Other nematodes, such as Trichostrongylus vitrinus, Trichostrongylus colubriformis, Nematodiurus battus, Teladorsagia circumcincta, Nippostrongylus brasiliensis and Haemonchus contortus have higher superoxide dismutase activities in the third larval stages of each species and showed marked interspecies variations in isoenzyme profiles (87).

5. NON-ENZYMATIC DEFENSES OF PARASITES AGAINST REACTIVE OXYGEN SPECIES Detoxication of reactive oxygen species is also possible through the action of several low-molecular-weight compounds. Some are soluble in water, such as ascorbic acid, thiols, pyruvate and urate, and some are lipid soluble, such as vitamin E and fl-carotone (1).

15 6

R. DOCAMPO

GSH is absent in some parasites such as E. histolytica (43), Tritrichomonasfoetus and Giardia duodenalis (44) where cysteine is the main thiol present. However, GSH is present in most parasites although its concentration varies widely. For example, it is low in trypanosomatids such as T. cruzi (88). In most trypanosomatids investigated thus far dihydrotrypanothione (T(SH2)) is present in concentrations of about 0.15 mM (48). T(SH2) is the principal intracellular thiol during exponential growth of C. fasciculata, whereas monoglutathionylspermidine is the major thiol in stationary phase. The net result of this biochemical reshuffle of metabolites can be described as follows: 2GSH-SPD ~ T(SH2) + spermidine which results in no net change in free GSH, but in the release of unconjugated spermidine. It has been suggested that the function of the redistribution of GSH-SPD into T(SH2) may be to provide higher levels of spermidine necessary for growth (89). T(SH2) has a standard redox potential of E': -0.242 + 0.002 V (59) which is marginally more negative than glutathione ( - 0 . 2 3 0 V) but considerably less than other physiological dithiols such as lipoic acid and thioredoxin. A role for pyruvate as H 2 0 2 scavenger in trypanosomes has been postulated (90). However, results indicate that the H202-decomposing activity of pyruvate in T. brucei is negligible (17). The presence of ascorbate peroxidase (32) in addition to a dehydroascorbate reductase in T. cruzi (91) has led to the suggestion of the occurrence of an enzymatic vitamin C redox cycle that would replenish ascorbic acid for reactive oxygen species removal (91). Trypanosomatids, as well as G. lamblia and Trichomonas spp. lack xanthine oxidase and therefore uric acid, another free radical scavenger, is absent. In contrast, a xanthine oxidase activity has been reported in Ancylostoma ceylanicum and Nippostrongylus brasiliensis (37). There are no reports of the presence of vitamin E and fl-carotene in parasites.

6. ACKNOWLEDGEMENTS Work from the author received financial support from the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases.

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30. Sanchez-Moreno, M., Lasztity, D., Coppens, I. and Opperdoes, F. R. (1992) Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias. Mol. Biochem. Parasitol. 54, 185-200. 31. Kulda, J., Kralova, J. and Vavra, J. (1977) The ultrastructure and 3,Y-diaminobenzidine cytochemistry of hydrogenosomes of trichomonads (Abstr). J. Protozool. 24: 51A. 32. Docampo, R., Boiso, J. F. de, Boveris, A. and Stoppani, A. O. M. (1976) Localization of peroxidase activity in Trypanosoma cruzi microbodies. Experientia 32: 972-975. 33. Bayne, R. A., Muse, K. E. and Roberts, J. F. (1969) Isolation of bodies containing the cyanide insensitive glycerophosphate oxidase of Trypanosoma equiperdum. Comp. Biochem. Physiol. 30: 1049-1054. 34. Michalski, W. P. and Prowse, S. J. (1991) Superoxide dismutases in Eimeria tenella. Mol. Biochem. Parasitol. 47:189-196. 35. Fairfield, A. S., Eaton, J. W. and Meshnick, S. R. (1986) Superoxide dismutase and catalase in the murine malaria, Plasmodium berghei: content and subcellular distribution. Arch. Biochem. Biophys. 250: 526-529. 36. Murray, H. W., Nathan, C. F. and Cohn, Z. A. (1980) Macrophage oxygen-dependent antimicrobial activity. IV. Role of endogenous scavengers of oxygen intermediates. J. Exp. Med. 152: 1610-1624. 37. Batra, S., Singh, S. P., Katiyar, J. C. and Srivastava, V. M. L. (1990) Reactive oxygen intermediates metabolizing enzymes in Ancyclostoma ceylanicum and N ipppostrongylus brasiliensis. Free Rad. Biol. Med. 8: 271-274. 38. Kazura, J. W. and Meshnick, S. R. (1984) Scavenger enzymes and resistance to oxygen mediated damage in Trichinella spiralis. Mol. Biochem. Parasitol. 10: 1-10. 39. Barrett, J. (1980) Peroxide metabolism in the liver fluke Fasciola hepatica. J. Parasitol. 66: 697. 40. Preston, C. M. and Barrett, J. (1987) Heligomosomoides polygyrus: peroxidase activity. Exp. Parasitol. 64: 24-28. 41. Campbell, T., Rubin, N. and Komuniecki, R. (1989) Succinate-dependent energy generation in Ascaris suum mitochondria. Mol. Biochem. Parasitol. 33: 1-12. 42. Chance, B., Sies, H. and Boveris, A. (1979) Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59: 527-605. 43. Fahey, R. C., Newton, G. L., Arrick, B., Overdank-Bogart, T. and Aley, S. B. (1984) Entamoeba histolytica: a eukaryotic without glutathione metabolism. Science 224: 70-72. 44. Brown, D. M., Upcroft, J. A. and Upcroft, P. (1993) Cysteine is the major low-molecular weight thiol of Giardia duodenalis. Mol. Biochem. Parasitol. 61: 155-158. 45. Thong, K. W. and Coombs, G. H. (1987) Comparative study of ferredoxin-linked and oxygen-metabolizing enzymes of trichomonads. Comp. Biochem. Physiol. 87B: 637-642. 46. Smith, N. C., Bryant, C. and Boreham, P. F. L. (1988) Possible roles for pyruvate:ferredoxin oxidoreductase and thiol-dependent peroxidase and reductase activities in resistance to nitroheterocyclic drugs in Giardia intestinalis. Int. J. Parasitol. 18: 991-997. 47. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. and Cerami, A. (1985) Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science 227: 1485-1487. 48. Henderson, G., Fairlamb, A. H. and Cerami, A. (1987) Trypanothione dependent peroxide metabolism in Crithidia fasciculata and Trypanosoma brucei. Mol. Biochem. Parasitol. 24: 39-45. 49. Carnieri, E. G. S., Moreno, S. N. J. and Docampo, R. (1993) Trypanothione-dependent peroxide metabolism in Trypanosoma cruzi different stages. Mol. Biochem. Parasitol. 61: 79-86. 50. Shames, S. L., Fairlamb, A. H., Cerami, A. and Walsh, C. T. (1986) Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases. Biochemistry 25: 3519-3526. 51. Krauth-Siegel, R. L., Enders, B., Henderson, G. B., Fairlamb, A. H. and Schirmer, R. H. (1987) Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme. Eur. J. Biochem. 164: 123-128.

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52. Hunter, W. N., Smith, K., Derewenda, Z. et al. (1990) Initiating a crystallographic study of trypanothione reductase. J. Mol. Biol. 216: 235-237. 53. Kuriyan, J., Kong, X.-P., Krishna, T. S. R. et al. (1991) X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4A resolution. Proc. Natl. Acad. Sci. USA 88: 8764-8768. 54. Bailey, S., Smith, K., Fairlamb, A. H. and Hunter, W. N. (1993) Substrate interactions between trypanothione reductase and Nl-glutathionylspermidine disulphide at 0.28-nm resolution. Eur. J. Biochem. 213: 67-75. 55. Zhang, Y., Bailey, S., Naismith, J. H. et al. (1993) Trypanosoma cruzi trypanothione reductase. Crystallization, unit cell dimensions and structure resolution. J. Mol. Biol. 233: 1217-1220. 56. Krauth-Siegel, R. L., Sticherling, C., Jost, I. et al. (1993) Crystallization and preliminary crystallographic analysis of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. FEBS Lett. 317: 105-108. 57. Lantwin, C. B., Schlichting, I., Kabsch, W., Pai, E. F. and Krauth-Siegel, R. L. (1994) The structure of Trypanosoma cruzi trypanothione reductase in the oxidized and NADPH reduced state. Proteins 18:161-173. 58. Smith, K., Opperdoes, F. R. and Fairlamb, A. H. (1991) Subcellular distribution of trypanothione reductase in bloodstream and procyclic forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 48: 109-112. 59. Fairlamb, A. H. and Henderson, G. (1987) Metabolism of trypanothione and glutathionylspermidine in trypanosomes. In: Host-Parasite Molecular Recognition and Interaction in Protozoal Infections (eds Chang, K. P. and Snary, D.), NATO ASI series, pp. 29-45. 60. Sullivan, F. X., Shames, S. L. and Walsh, C. T. (1989) Expression of Trypanosoma congolense trypanothione reductase in Escherichia coli: overproduction, purification and characterization. Biochemistry 28: 4986-4992. 61. Sullivan, F. X. and Walsh, C. T. (1991) Cloning, sequencing, overproduction and purification of trypanothione reductase from Trypanosoma cruzi. Mol. Biochem. Parasitol. 44: 145-148. 62. Field, H., Cerami, A. and Henderson, G. B. (1992) Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata. Mol. Biochem. Parasitol. 50: 47-56. 63. Aboagye-Kwarteng, T., Smith, K. and Fairlamb, A. H. (1992) Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic mechanism. Mol. Microbiol. 6: 3089-3099. 64. Henderson, G., Murgolo, N. J., Kuriyan, J. et al. (1991) Engineering the substrate specificity of glutathione reductase toward that of trypanothione reductase. Proc. Natl. Acad. Sci. USA 88: 8769-8773. 65. Fairlamb, A. H. and Cerami, A. (1985) Identification of a novel, thiol-containing co-factor essential for glutathione reductase enzyme activity in trypanosomatids. Mol. Biochem. Parasitol. 14: 187-198. 66. Penketh, P. G., Kennedy, W. P. K., Patton, C. L. and SartoreUi, A. C. (1987) Trypanosomatid hydrogen peroxide metabolism. FEBS Lett. 221: 427-431. 67. Kelly, J. M., Taylor, M. C., Smith, K., Hunter, K. J. and Fairlamb, A. H. (1993) Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over-express trypanothione reductase. Sensitivity towards agents that are thought to induce oxidative stress. Eur. J. Biochem. 218: 29-37. 67. Meshnick, S. R. and Eaton, J. W. (1981) Leishmanial superoxide dismutase: a possible target for chemotherapy. Biochem. Biophys. Res. Commun. 102: 970-976. 68. Fritsch, B., Dieckmann, A., Menz, B. et al. (1987) Glutathione and peroxide metabolism in malaria-parasitized erythrocytes. Parasitol. Res. 73:515- 517. 69. Hempelmann, E., Heiner-Schirmer, R., Fritsch, G., Hundt, E. and Groschel-Stewart, U. (1987) Studies on glutathione reductase and methemoglobin from human erythrocytes parasigzed with Plasmodium falciparum. Mol. Bioehem. Parasitol. 23: 19-24. 70. Williams, D. L., Pierce, R. J., Cookson, E. and Capron, A. (1992) Molecular cloning and sequencing of glutathione peroxidase from Schistosoma mansoni. Mol. Biochem. Parasitol. 52: 127-130.

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71. Tannich, E., Bruchhaus, I., Walter, R. and Horstmann, R. D. (1991) Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an ironcontaining superoxide dismutase. Mol. Biochem. Parasitol. 49: 61-72. 72. Lindmark, D. G. and Miiller, M. (1974) Superoxide dismutase in the anaerobic flagellates Tritrichomonas foetus and Monocercomonas sp. J. Biol. Chem. 249: 4634-4637. 73. Kitchener, K. R., Meshnick, S. R., Fairfield, A. S. and Wang, C. C. (1984) An iron-containing superoxide dismutase in Tritrichomonasfoetus. Mol. Biochem. Parasitol. 12: 95-99. 74. Boveris, A. and Stoppani, A. O. M. (1977) Hydrogen peroxide generation in Trypanosoma cruzi. Experientia 33:1306-1308. 75. Asada, K., Kanematsu, S., Okada, S. and Hayakawa, T. (1980) Phylogenetic distribution of three types of superoxide dismutase in organisms and cell organelles. In: Chemical and Biochemical Aspects of Superoxide and Superoxide Dismutases (eds Bannister, J. V. and Hill, H.), Elsevier, New York, pp. 136-153. 76. Opperdoes, F. R., Borst, P., Bakker, S. and Leene, W. (1977) Localization of glycerol3-phosphate oxidase in the mitochondrion and particulate NAD +-linked glycerol-3-phosphate dehydrogenase in the microbodies of the bloodstream form of Trypanosoma brucei. Eur. J. Biochem. 76: 29-39. 77. Le Trang, N., Meshnick, S. R., Kitchener, K., Eaton, J. W. and Cerami, A. (1983) Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to leishmanial and trypanosomal enzymes. J. Biol. Chem. 258: 125-130. 78. Fairfield, A. S., Meshnick, S. R. and Eaton, J. W. (1983) Malaria parasites adopt host cell superoxide dismutase. Science 221: 764-766. 79. Ranz, A. and Meshnick, S. R. (1989) Plasmodium falciparum: inhibitor sensitivity of an endogenous superoxide dismutase. Exp. Parasitol. 69: 125-128. 80. Becuwe, P., Slomianny, C., Valentin, A., Schrevel, J., Camus, D. and Dive, D. (1992) Endogenous superoxide dismutase activity in two Babesia species. Parasitolooy 105:177-182. 81. Sidbley, L. D., Lawson, R. and Weidner, E. (1986) Superoxide dismutase and catalase in Toxoplasma 9ondii. Mol. Biochem. Parasitol. 19: 83-87. 82. Simurda, M. C., van Keulen, H., Rekosh, D. M. and LoVerde, P. T. (1988) Schistosoma mansoni: identification and analysis of an mRNA and a gene encoding superoxide dismutase. Exp. Parasitol. 67: 73-84. 83. Cordeiro da Silva, A., LePresle, T., Capron, A. and Pierce, R. J. (1992) Molecular cloning of a 16-kilodalton Cu/Zn superoxide dismutase from Schistosoma mansoni. Mol. Biochem. Parasitol. 52: 275-278. 84. Leid, W. R. and Suquet, C. M. (1986) A superoxide dismutase of metacestodes of Taenia taeniformis. Mol. Biochem. Parasitol. 18: 301-311. 85. Rhoads, M. L. (1983) Trichinella spiralis: identification and purification of superoxide dismutase. Exp. Parasitol. 56: 41-45. 86. Henkle, K. L., Liebau, E., Miiller, S., Bergmann, B. and Walter, R. D. (1991) Characterization and molecular cloning of a Cu/Zn superoxide dismutase from the human parasite Onchocerca volvulus. Infect. lmmun. 59: 2063-2069. 87. Knox, D. P. and Jones, D. G. (1992) A comparison of superoxide dismutase (SOD, EC 1.15.1.1) distribution in gastro-intestinal nematodes. Int. J. Parasitol. 22: 209-214. 88. Boveris, A., Sies, H., Martino, E. E., Docampo, R., Turrens, J. F. and Stoppani, A. O. M. (1980) Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem. J. 188: 643-648. 89. Shim, H. and Fairlamb, A. H. (1988) Levels of polyamines, glutathione, and glutathionespermidine conjugates during growth of the insect trypanosomatid Crithida fasciculata. J. Gen. Microbiol. 134: 807-817. 90. Fulton, J. and Spooner, J. (1956) Inhibition of the respiration of Trypanosoma rhodesiense by thiols. Biochem. J. 63: 475-482. 91. Clarke, D., Albrecht, M. and Ar6valo, J. (1994) Ascorbate variations and dehydroascorbate reductase activity in Trypanosoma cruzi epimastigotes and trypomastigotes. Mol. Biochem. Parasitol. 66: 143-145.

10 Xenobiotic Metabolism J A M E S W. T R A C Y ~ and ELIZABETH A. V A N D E W A A 2

~Departments of Comparative Biosciences and Pharmacology and Environmental Toxicology Center and 2Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, Wl, USA

SUMMARY

Over the past decade, work from several laboratories has demonstrated that parasites can metabolize xenobiotics (foreign chemicals), including some antiparasitic drugs. In the case of the nitroheterocyclic agents metronidazole, nifurtimox, and niridazole, enzymatic nitroreduction within the target organism is responsible for the antiparasitic action of these drugs. Alternatively, xenobiotic metabolism can result in inactivation or detoxication of potentially toxic xenobiotics. For example, glutathione S-transferase-catalysed conjugation of electrophilic chemicals with glutathione has been found to play a role in cellular protection in several different parasites. Despite these few well-characterized examples, however, relatively little is known about the capacity of most parasites to metabolize xenobiotics. This chapter surveys the known pathways of xenobiotic metabolism in helminth and protozoan parasites and attempts to highlight areas where further investigation could prove useful. Elucidating the pathways by which parasites metabolize xenobiotics could provide important insights into parasite-drug interactions that may reveal novel strategies for rational drug design.

1. INTRODUCTION

Both free-living and parasitic organisms are exposed to xenobiotics (foreign chemicals) in their environment. Many of these chemicals, which by definition are not required for normal metabolic functions, are lipophilic and are readily absorbed into cells by passive Copyright 9 1995 Academic Press Ltd All rights of reproduction in any form reserved

Biochemistry and Molecular Biology of Parasites ISBN 0-12-473345-X

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non-ionic diffusion. A few xenobiotics are eliminated unchanged and some undergo spontaneous chemical rearrangement or decomposition. The vast majority, however, undergo enzyme-catalyzed biotransformation into metabolites. These metabolites are generally more water soluble than the present compound and are more readily eliminated from the cell. Thus, xenobiotic metabolism is usually synonymous with detoxication. There are exceptions to this rule. Sometimes xenobiotics are metabolized to chemically reactive products that are toxic. The pathways of xenobiotic metabolism can be divided into two groups of reactions. Phase I metabolism includes oxidation, reduction and hydrolysis. These are sometimes called 'functionalization' reactions, because a polar functional group ( e . g . , - - O H , - - C O O H , w N H 2 , etc.) is introduced into the molecule. Phase II metabolism includes various synthetic reactions between a polar functional group on a xenobiotic substrate and an endogenous cosubstrate. Because this results in formation of a new covalent bond, these reactions are collectively referred to as 'conjugation reactions' and the reaction products as 'conjugates'. Often the substrate of a phase II reaction is a phase I metabolite. The net result is usually an increase in the water solubility of the xenobiotic, which again favors its elimination from the cell. A detailed treatment of xenobiotic metabolism pathways can be found in monographs by Jakoby (1,2) and Mulder (3). The chemistry of xenobiotic metabolism is covered in works by Jakoby et al. (4) and Anders (5). In contrast to the information available on carbohydrate and energy metabolism in parasites, relatively little is known about the fate of xenobiotics, including clinically used antiparasitic drugs. This chapter deals primarily with xenobiotic metabolism in helminths, although where available, information on the fate of foreign compounds in protozoan parasites is also included.

2. PHASE I METABOLISM 2.1. Oxidation Reactions 2.1.1. Cytochromes P450

In most organisms, the prototypic phase I oxidation reaction is catalyzed by a family of microsomal hemoproteins, the cytochromes P450 (6). These enzymes are widely distributed in nature being found in vertebrates, insects, bacteria and plants. In parasites, however, the only cytochrome P450 detected thus far has been that in Trypanosoma cruzi epimastigotes (7). It was speculated that T. cruzi might utilize a cytochrome P450 system to metabolize foreign compounds, rendering this parasite insensitive to chemotherapeutic attack (7). However, that hypothesis has not been rigorously tested. The apparent absence of cytochromes P450 from all other parasite species examined is therefore particularly enigmatic. A number of explanations have been proposed, but none is entirely convincing. These have been reviewed by Precious and Barrett (8). One caveat should be borne in mind. In all cases, the methods used to measure either cytochrome P450 content or catalytic activity have been relatively insensitive. Although mammalian liver is the richest source of cytochromes P450, there are now several

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examples of extrahepatic tissues that contain very low levels of cytochromes P450 which are nevertheless important in xenobiotic metabolism (9). Thus, it remains to be seen whether the apparent absence of cytochromes P450 in most parasites is merely due to the limitations of the methods used to detect them. The use of highly conserved cytochromes P450 gene sequences (6) to search parasite genomes for this important group of xenobiotic metabolizing enzymes is another approach that has yet to be exploited. 2.1.2. Sulfoxidase

A few other oxidation reactions have been reported in helminths. The cytosolic fractions of Moniezia expansa and Ascaris suum catalyze the sulfoxidation of several xenobiotics, including the benzimidazole derivatives albendazole and fenbendazole, to the corresponding sulfoxides (10). The reaction requires NADPH and molecular oxygen, but unlike mammalian sulfoxidase, it is not associated with the microsomal fraction. Although parasite sulfoxidases have not been purified, their properties suggest they are soluble forms of flavin-linked monooxygenase. 2.1.3. Peroxidases

Glutathione (GSH) peroxidase (EC 1.11.1.9) is one of several selenoproteins that contain a unique selenocysteine residue at the active site (1). This enzyme catalyzes the GSH-dependent reduction of both hydrogen peroxide and organic hydroperoxides, including fatty acid hydroperoxides formed during lipid peroxidation. GSH peroxidase activity has been measured in both protozoan and helminth parasites (11,12). The enzyme from Schistosoma mansoni has been cloned (13). The presence of the unique selenocysteine codon (UGA) confirms that it is a selenoprotein. Although GSH peroxidase activity appears to be absent from Hymenolepsis diminuta and M. expansa (11), these parasites do contain a peroxidase activity capable of oxidizing cytochrome c (cytochrome c peroxidase) (14). The latter enzyme also is present in S. mansoni (15), but its role in xenobiotic metabolism has yet to be established. Additional information regarding parasite peroxidases can be found in Chapter 9. 2.1.4. Xanthine oxidase

Xanthine oxidase (EC 1.2.3.2) catalyzes the formation of uric acid, an end-product of purine catabolism. The mammalian enzyme is a metalloflavoprotein composed of two subunits containing molybdenum, FAD and Fe/S clusters as prosthetic groups in a ratio of 1:1:4 per subunit (1). Besides its endogenous metabolic function, xanthine oxidase is also active toward a wide spectrum of oxidizable xenobiotic substrates. Although some cestodes and trematodes produce trace amounts of uric acid (16), the presence of xanthine oxidase activity in these organisms has not been demonstrated. Xanthine oxidase was found in the cytosolic fractions of the nematodes Ancylostoma ceylanicum and Nippostrongylus brasiliensis (17), but its activity toward xenobiotic substrates was not tested. The role of xanthine oxidase in the generation of superoxide radical as a reaction product is often cited, and a xanthine-xanthine oxidase mixture is a convenient way

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to generate superoxide in vitro as a means of testing a parasite's antioxidant capacity (11). It should be noted that under normal conditions, xanthine oxidase is a dehydrogenase. Conversion of the dehydrogenase form into the oxidase often occurs inadvertently during enzyme purification. In mammalian tissues, prolonged ischemia causes the dehydrogenase to be converted into the oxidase form (18). Under that circumstance, superoxide radical is produced and extensive tissue damage may result. Whether an analogous process takes place in living parasites is unknown. 2.1.5. Monoamine oxidase

Monoamine oxidase (EC 1.4.3.4) activity is present in homogenates of adult S. mansoni (19). This flavoprotein, which catalyzes the oxidative deamination of amines, functions in the breakdown of endogenously produced neurotransmitter amines. Vertebrate monoamine oxidase also catalyzes the oxidation of xenobiotic amines, particularly in tissues such as the intestine (4). Whether the schistosome enzyme is also capable of metabolizing xenobiotics has not been investigated. 2.2. Reduction Reactions Although oxidation reactions in parasites are relatively uncommon, there are several examples of parasite enzymes that catalyze the reductive metabolism of xenobiotics. 2.2.1. Azoreductase and nitroreductase

Helminth parasites have the capacity to reductively metabolize a variety of xenobiotics. Whereas all helminth species examined are able to reduce xenobiotics containing an azo functional group (R--N---N--R'), nitroreduction is not universally present. For example, A. suum and M. expansa reduce both azobenzene and 4-nitrobenzoic acid (20), whereas H. diminuta (21) and Onchocerca gutturosa (22) metabolize only the former. Although azoreductase and nitroreductase have not been purified from helminths, they appear to be confined to the soluble cell fraction and to use NADH, rather than NADPH, as the reduced pyridine cofactor (8). An exception is the nitroreductase from S. mansoni which displays greater activity with NADPH compared with NADH (23). A number of protozoan parasites including Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and T. cruzi also reductively metabolize xenobiotics containing a nitro functional group (24). 2.2.2. Role of nitroreduction & the metabol&m and mode of action of nitroheterocyclic antiparasitic drugs

A good illustration of how xenobiotic metabolism can result in toxic activation rather than detoxication is the role of parasite enzymes in the biotransformation of nitroheterocyclic drugs. Metronidazole, a 5-nitroimidazole derivative, is used for the treatment of T. vaginalis infections (25). The drug is reduced by trichomonads to form a nitroradical anion that can be demonstrated by electron spin resonance spectroscopy (24). The reaction is catalyzed by pyruvate:ferredoxin oxidoreductase localized in the hydrogenosome, an organelle unique to trichomonads (see Chapter 3). Under physiological conditions, metronidazole undergoes further metabolism in trichomonads to

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other reduced metabolites (nitroso and hydroxylamino derivatives) with eventual ring opening to yield acetamide and probably N-(2-hydroxyethyl)oxamic acid (26). The nitroradical anion does not react with cellular nucleophiles, but [14C]metronidazole does bind covalently to proteins and nucleic acids in T. foetus (26). This is consistent with further reduction of the nitroradical anion. The interaction of these reactive intermediates with macromolecules is probably responsible for the ultimate cytotoxic action of metronidazole in trichomonads. Under aerobic conditions, redox cycling of metronidazole occurs leading to formation of superoxide radical (24). In T. foetus, reoxidation of the nitroradical with concomitant formation of superoxide has been suggested as a detoxication pathway to protect the organism from metabolites resulting from further reduction of the nitro radical anion. The superoxide radical and other reactive oxygen metabolites formed are then detoxicated by enzymes involved in protection against oxidative stress (see Chapter 9). Metronidazole is also effective against the anaerobic intestinal parasites E. histolytica and G. lamblia. Although the metabolic fate of the drug has not been studied in these species, its mechanism of action also is likely due to proximal nitroreduction. Nifurtimox, a 5-nitrofuran derivative, is effective against T. cruzi infections. As with metronidazole, nifurtimox undergoes proximal nitroreduction within the target organism to form the nitroradical anion. Unlike the previous example, however, the toxicity of nifurtimox appears due to reactive oxygen metabolites formed as a result of nifurtimox redox cycling (24). Unlike trichomonads, T. cruzi is characterized by a deficiency in its enzymatic defenses against reactive oxygen species (see Chapter 9). The antischistosomal drug niridazole, a 5-nitrothiazole derivative, undergoes enzyme-catalyzed nitroreduction within adult S. mansoni (23). In contrast to the situation with nifurtimox in T. cruzi, redox cycling with resulting formation of reactive oxygen metabolites appears to play a negligible role in the action of niridazole. In this case, the nitro radical anion undergoes further reduction by dismutation of the radical to form other reactive metabolites such as the nitroso (--N---O) and hydroxylamino ( - - N H - - O H ) derivatives that react with cellular nucleophiles (GSH, proteins, nucleic acids) to form covalent adducts. Ring opening of the hydroxylamino derivative takes place leading to formation of the immunosuppressive metabolite 1-thiocarbamoyl-2imidazolidinone (27). This metabolite, however, has no antischistosomal activity. Drug disposition studies using [14C]niridazole demonstrated that over 90% of the niridazole absorbed by S. mansoni is extensively metabolized (23). Nearly 30% of the radioactivity absorbed by the parasite was found bound to macromolecules and about 50% was present as unidentified water-soluble metabolites. Metabolism of niridazole in schistosomes is quite distinct from that in the mammalian host, where the primary route of biotransformation involves cytochrome P450-catalyzed oxidation of the 2-imidazolidinone ring (28). Significantly, no oxidative metabolites are formed in schistosomes (23), consistent with the general view that these parasites are deficient in oxidative drug metabolism. That the metabolism of niridazole involved reduction to a nitro radical was supported by the observation that drug metabolism in cell-flee extracts of S. mansoni was inhibited by molecular oxygen, but that consumption of reduced pyridine nucleotide was stimulated by niridazole. Although this is consistent with redox cycling of the nitro radical in the presence of oxygen, net reduction occurs in intact parasites even when they are incubated under aerobic conditions (23). That nitroreduction and subsequent covalent binding to parasite nucleophiles is involved in the activation of niridazole was demonstrated using

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the 4'-methyl analog. 4'-methyl[14C]niridazole, which has no antischistosomal activity, was readily absorbed by schistosomes but was not metabolized and was not a substrate for schistosome nitroreductase (23). The exact metabolic fate of a particular nitroheterocycle is dependent on both its redox potential and the nature of the nitroreductase found in a given parasite species. Furthermore, it is difficult to identify the sequence of secondary biochemical events that ultimately leads to parasite death. However, in each of the cases cited above, enzyme catalyzed proximal nitroreduction is an obligatory step in the metabolic activation and subsequent antiparasitic action of these drugs. 2.2.3. Other reduction reactions

Aldehyde reductases and ketone reductases are families of enzymes involved in reductive metabolism of both endogenously produced and xenobiotic aldehydes and ketones (1). Aldehyde reductase activity has been detected in cell-free preparations of H. diminuta, Heliymosomoides polyoyrus and in the free-living nematode Panaorellus redivivus (8). Ketone reductase activity, on the other hand, appears absent from these organisms. T. cruzi contains NADPH-cytochrome c reductase activity in both the microsomal and cytosolic-cell fractions (29). The cytosolic enzyme may act as an oxygenase, protecting the cell in times of hydrogen peroxide production, since trypanosomes are deficient in catalase (30). It may also metabolize drugs directly, possible conferring resistance to antitrypanosomal drugs (31). The particulate enzyme form probably functions in microsomal electron transport associated with trypanosomal cytochrome P450. Despite the fact that quinones are ubiquitous in nature and many of them are cytotoxic (32), little is known of their metabolic fate in parasites. The cellular reduction of quinones proceeds via a one- or two-electron transfer catalyzed by a variety of flavoproteins. One-electron reduction to the semiquinone radical (typically catalyzed by NADPH-cytochrome P450 reductase or NADH-ubiquinone oxidoreductase) can initiate redox cycling and oxidative stress. In contrast, two-electron reduction (catalyzed by NAD(P)H:quinone oxidoreductase or DT-diaphorase) to form the corresponding hydroquinone is often viewed as a detoxication reaction (32). Quinone reductase activity has been reported in trypanosomes (12), and two distinct diaphorase activities have been detected in E. histolytica (33). Unlike the mammalian enzyme, however, the protozoan enzyme was insensitive to dicoumarol, a highly specific DT-diaphorase inhibitor. Bueding (34) observed that menadione (2-methyl-l,4-naphthoquinone) was toxic to S. mansoni. It interfered with glucose metabolism and its effect was potentiated under aerobic conditions. Although this observation is consistent with the known redox cycling of menadione, the metabolism of that compound has not been investigated in schistosomes.

2.3. Hydrolysis Reactions Parasites have the enzymatic capacity to hydrolyze a variety of xenobiotics. M. expansa, A. suum and H. diminuta contain O- and N-deacetylase activity, aryl sulfatase, and both acid and alkaline phosphatases (16,21). The cestode H. diminuta can hydrolyze both ~-

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and fl- glycosidic linkages. Esterase activity in these organisms can hydrolyze a number of model substrates. Significantly, however, helminths appear to lack the capacity to hydrolyze fl-glucuronide substrates, indicating the absence of fl-glucuronidase activity. S. mansoni contains esterase activity both toward the endogenous neurotransmitter acetylcholine and toward organophosphorus compounds such as metrifonate (35). Epoxides are one class of electrophilic xenobiotics that are often toxic (4). Hydrolysis of the strained epoxide ring is catalyzed by epoxide hydrolase (EC 3.3.2.3) to give the corresponding trans-dihydrodiol that is not electrophilically active. Cell-free preparations of O. gutturosa contained low levels of epoxide hydrolase activity toward trans-stilbene oxide (22), but no activity was detected in H. diminuta (21). Since epoxide hydrolase in O. gutturosa was detected in a preparation containing both microsomes and cytosol, it is impossible to determine whether the enzyme is among the common microsomal epoxide hydrolases or whether it is cytosolic. Microsomal fractions prepared from T. cruzi epimastigotes showed epoxide hydrolase activity toward styrene oxide (36), which was increased three-fold when the organisms were exposed to the inducing agent phenobarbital. A more extensive survey would be useful to determine how common epoxide hydrolase is among protozoan and helminth parasites.

3. PHASE II METABOLISM 3.1. Conjugation with Glutathione Conjugation of lipophilic compounds bearing an electrophilic center with the endogenous nucleophilic glutathione (GSH) is a major pathway of xenobiotic metabolism in most organisms (5, 37). Of all xenobiotic metabolism pathways characterized to date, conjugation with GSH is by far the most prevalent among both helminth and protozoan species. These reactions are catalyzed by a multifunctional family of mostly cytoplasmic enzymes, the GSH S-transferases (EC 2.5.1.18). These enzymes are highly specific for the endogenous cosubstrate GSH, but bind a broad spectrum of xenobiotics and endogenously produced electrophiles by virtue of a less-specific hydrophobic region at the active site (37). GSH S-transferases are widely distributed in animals, being found in both vertebrates (37) and non-vertebrates (38), including helminth and protozoan parasites. Brophy and Barrett (39) have reviewed the distribution of GSH S-transferases in helminths and have observed that digean trematodes and intestinal cestodes generally have higher levels of total GSH S-transferase activity than do nematodes. Of the helminths examined to data, schistosomes show the highest level of activity, some 5-50-fold higher than others (40). GSH S-transferases have been identified and characterized from several protozoan species, including T. cruzi (41) and several Acanthamoeba species (42). No activity was detected in E. histolytica, G. intestinalis or T. vaginalis (42). The observation that E. histolytica lacks GSH S-transferase is consistent with the lack of GSH and its associated pathways in this organism (24). It is particularly curious that T. cruzi contains a typical GSH S-transferase, since trypanothione, rather than GSH, is the major intracellular thiol in these organisms (see Chapter 9).

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Many organisms contain multiple forms of GSH S-transferase, but the isoenzymes of mammalian liver have been the most extensively studied (37). These abundant enzymes are characterized by distinct but overlapping substrate specificities that account for the liver's capacity to conjugate a structurally diverse group of chemicals. Based on primary structure, physicochemical properties and catalytic specificities, mammalian GSH S-transferases have been assigned to one of three classes (~, # or n) that correspond three major gene families (43). The catalytically active enzymes exist as binary combinations of either identical (homodimer) or certain combinations of non-identical subunits (heterodimer); but heterodimers do not form between subunits of different classes (43). The best characterized parasite GSH S-transferases are those of schistosomes. There are at least six biochemically distinct forms of the enzyme in adult S. mansoni. The three major isoenzymes (SmGST-1, SmGST-2 and SmGST-3), which comprise 2-4% of the total soluble protein (40), form an isoenzyme family composed of two hornodimers (SmGST-1 and SmGST-3) and the corresponding heterodimer (SmGST-2) (44). Further evidence that SmGST-1 and SmGST-3 are closely related gene products comes from observations that an SmGST-3 cDNA hybridizes to messages encoding both subunits (45). Moreover, the ratio of two 28 kDa SmGST sequence variants in a cDNA library and the deduced isoelectric points of the encoded proteins (46) are consistent with the isoelectric points of SmGST-1 and SmGST-3 (40) and the ratio of the corresponding messages (45). From a comparison of its abundance, catalytic and physicochemical properties, SmGST-3 (subunit M r - 28 500) is equivalent to the isoenzyme characterized as P28 and Sm28, which is being investigated as a candidate vaccine antigen (39, 47). The P28 GSH S-transferase was reported to be a surface antigen (47). However, a separate study failed to detect any GSH S-transferase within or on the tegument, including the dorsal spines of the male worm (48). Instead the enzymes diaplayed a punctate pattern of distribution that was restricted to the parenchyma (48). Besides the SmGST-1/2/3 isoenzyme family, at least three other catalytically active SmGSTs are present in adult S. mansoni. SmGST-4 (subunit M r - 26000), can be distinguished from the SmGST-1/2/3 family by its physicochemical properties, catalytic specificity, and its selective elution from GSH-agarose affinity columns with glutathione disulfide (49). SmGST-5 is a labile form of the enzyme that does not bind to GSH-agarose (40). It preferentially catalyzes the conjugation of epoxide substrates and the organophosphorus compound dichlorvos (50). Finally, SmGST-6 (subunit M r = 28 000) is an enzyme form that binds very weakly to GSH-agarose, with most of the activity being found in the unbound fraction. Unlike SmSGT-5, however, this isoenzyme is quite stable (Tracy and Vande Waa, unpublished observation). cDNAs encoding several schistosome GSH S-transferases have been isolated (39, 45, 51, 52). Attempts have been made to include schistosome GSH S-transferases under the ct, #, n classification system (37). Although some short stretches of sequence similarity exist, it is clear that parasite and mammalian GSH S-transferases are at best distantly related (53). This difference is not too surprising in light of the ability of schistosome GSH S-transferases to stimulate host immunity. Besides catalyzing the conjugation of numerous model xenobiotics, parasite GSH S-transferases are implicated in resistance to certain antiparasitic drugs. One form of S. mansoni GSH S-transferase (SmGST-5) catalyzes the GSH-dependent demethylation of dichlorvos, the active form of the antischistosomal drug metrifonate (50). The two

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metabolites that form, S- methylglutathione and des-methyldichlorvos are both pharmacologically inactive. The level of GSH S-transferase activity toward the model substrate 1-chloro-2,4-dinitrobenzene in the nematode H. contortus appears to correlate with resistance to cambendazole (39), although the conjugation of cambendazole was not demonstrated. GSH S-transferases can also play a role in protection against drug toxicity by acting as intracellular binding proteins (37). Several anthelmintics have been shown to bind to GSH S-transferases from M. expansa, H. diminuta and F. hepatica, although they do not conjugate these drugs (39). Those observations are consistent with the view that GSH S-transferases can participate in cellular protection by reducing the bioavailability of toxicants (43). Although most studies have focused on the role of GSH S-transferases in xenobiotic metabolism, these enzymes also have multiple endogenous metabolic functions. For example, the biosynthesis of leukotriene C 4, the GSH conjugate of the epoxide leukotriene A 4, was shown to be catalyzed by the GSH S-transferase from Dirofilaria immitis (54). In addition to the selenium-dependent GSH peroxidase, eukaryotic cells have selenium-independent GSH peroxidase activity that is attributable to GSH S-transferase (37). As such, GSH S-transferases play a role in the reduction of fatty acid hydroperoxides formed during lipid peroxidation. In contrast to selenium-dependent GSH peroxidase, GSH S-transferases do not catalyze the reduction of hydrogen peroxide. Several helminth GSH S-transferases have been shown to catalyze the reduction of lipid and organic hydroperoxides (39, 49). GSH S-transferases catalyze the detoxication of 4-hydroxyalk-2-enals (37,43). These ~,fl-unsaturated aldehydes are formed as byproducts of lipid peroxidation and are implicated as the ultimate cytotoxic products (55). The GSH S-transferases from S. mansoni catalyze the conjugation of a series of 4-hydroxyalk-2-enals, including 4-hydroxynon-2-enal, the isomer that is formed under physiological conditions (49). This observation supports the idea that these enzymes may play a role in protecting the adult parasites against host-mediated oxidative mechanisms. The expression of numerous xenobiotic metabolizing enzymes can be induced by exposing the organism to a number of xenobiotics. For example, GSH S-transferase expression can be induced in mammalian tissues by treatment with various compounds, including the barbiturate phenobarbital, planar aromatic hydrocarbon carcinogens such as 3-methylcholanthrene, the environmental toxicant 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD), phenolic antioxidants such as 2(3)-tert-butyl-4-hydroxyanisole (BHA), and Michael reaction acceptors that contain an electrophilic olefin such as ~, fl-unsaturated aldehydes and the antischistosomal drug oltipraz (37). Induction is characterized by an increase in enzyme content or specific activity and is paralleled by an increase in specific mRNA. Because not all GSH S-transferase subunits are induced by a given agent, only modest increases in total catalytic activity (0.SmM) and by a carrier-mediated process at lower concentrations. At lower concentrations, absorption of this lipid precursor is non-linear, dependent on temperature and pH, and competitively inhibited by glycerol and ~-glycerophosphate. The existence of two distinct carriers for this molecule is suggested by studies which show that only about half the saturable component in H. diminuta is Na§ and inhibitable by 1,2-propandiol (2). In H. diminuta, two distinct carrier-mediated transport systems for fatty acid absorption have been partially characterized, one specific for short-chain fatty acids and the other for longer chain molecules. Acetate absorption in H. diminuta is mediated by the short-chain carrier at low concentrations, but passive diffusion occurs at high concentrations (15). Amino acid absorption in adult cestodes occurs through multiple carriers that are saturable and sensitive to temperature and to a variety of inhibitors (2, 13). Amino acid accumulation typically occurs against steep concentration gradients (16), and the amino acid carrier systems in cestodes seem to be similar to those in mammals. There are separate carriers for acidic and basic amino acids and multiple carriers for the neutral amino acids, which overlap in their specificities. Unlike mammals, however, which show higher affinities for L-amino acids, cestode transporters are not stereoselective. Also,

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amino acid absorption in some cestodes is not dependent on co-transport of inorganic ions; replacement of Na + with choline or K + does not affect amino acid transport kinetics (16). The available evidence suggests that cestodes can synthesize pyrimidines de novo, whereas purines must be absorbed from host fluids across the tegument (2). Both types of nitrogenous base are absorbed in vitro, mediated in part in H. diminuta by at least three distinct carriers, two of which contain multiple substrate binding sites (17). A passive diffusional component contributes to both purine and pyrimidine absorption (Chapter 6). Endocytosis does not appear to play a major role in the absorption of proteins, though the capacity of cestodes to absorb macromolecules has not been clearly delineated. Horseradish peroxidase and ruthenium red appear to be endocytosed by the pseudophyllidean cestodes Lingula intestinalis and Schistocephalus solidus (18) and by Hymenolepis nana (19). The apparent lack of endocytosis detected in most studies that have addressed this topic may be attributable to the fact that only mature regions of the proglottids were examined (2).

2.1.2.2. Digestion. As in the epithelial lining of the vertebrate small intestine, the cestode tegument contains many enzymes usually associated with digestion, including alkaline and acid phosphatases, esterases and disacharidases (20,21). It is not known, however, if digestive enzymes associated with the outer tegumental membrane are derived from the parasite or the host. The concept that these enzymes may participate in extracorporeal digestion of nutrient macromolecules, i.e., 'contact digestion' (21), is a matter of controversy (22). Proteins within the glycocalyx and the membrane of the underlying microtriches are not digested by proteolytic enzymes in the lumen of the host intestine, and this may be due to a physical property of the glycocalyx. One possibility is that the glycocalyx helps maintain an unstirred water layer between the parasite and the contents of the host intestine. The organic acid end-products of carbohydrate metabolism excreted by cestodes could form a microenvironment in the immediate vicinity of the tegument that is too acidic for host proteases to function (23). Alternatively, the glycocalyx may contain factors, as yet undefined, that specifically inhibit host hydrolytic enzymes. For instance, H. diminuta inactivates trypsin and chymotrypsin by releasing a protease inhibitor (24). 2.1.2.3. Transport of inorganic ions. Homeostatic regulation of inorganic ion concentrations is a key function of all transport epithelia. Proteins that serve this function in vertebrates are also present in cestodes. The tegument contains a Ca 2 +-ATPase activity which, by analogy with better characterized systems, is essential for maintaining intracellular Ca 2 + at low concentrations. Two forms of the enzyme occur in the outer tegumental membrane of H. diminuta, one of which is calmodulin-dependent (25). Na+/K +-ATPase, a key enzyme in the regulation of intracellular levels of these cations in most organisms, has been demonstrated in the tegument of E. 9ranulosus, but has not yet been detected in other cestodes (3). The effect of ouabain, a specific inhibitor of this enzyme, on amino acid absorption by H. diminuta suggests that Na+/K +ATPase is present and functional in other species.

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2.1.2.4. Water balance and excretion. In addition to regulating essential electrical gradients across neuronal and muscle membranes, ion transport in cestodes, like other metazoans, may also be linked to the control of hydrostatic pressure gradients across cell membranes and across the body wall. Osmoconforming organisms behave as simple osmometers, and shrink or swell in direct relation to the osmolarity of their environment. Some cestodes are osmoconformers, adjusting internal osmotic pressure by varying fluid volume (2). A few species, including H. diminuta, regulate volume (i.e., maintain their weight at a constant level) over a fairly wide range of osmotic pressures; however, the worms rapidly swell or shrink in media which are below or above that range, respectively (23). How H. diminuta regulates its volume is not known, but the ability to excrete organic acid end-products of glucose metabolism (succinate and acetate) appears to be important. Osmoregulation in this cestode is glucose-dependent, and both glucose absorption and metabolism increase linearly with osmotic pressure of 210-335 mosmol/1, whereas the rates of uptake of leucine, Na + and C1- are unaffected by osmotic pressure. The high levels of succinate and acetate excreted as a consequence of osmotically induced glucose metabolism may acidify the medium surrounding the parasite (23, 26).

3. TREMATODES Unlike cestodes, trematodes have an internal surface, the gastrovascular cavity (gastrodermis), which has a single opening that serves as both mouth and anus. The gastrodermis is likely to be an important organ for nutrient digestion and absorption, though definitive experimental support is lacking. The belief that tegument-associated antigens are the best candidates for vaccine development has motivated considerable research on tegument proteins and the effects of immune substances on the external surface, primarily in Schistosoma spp. Few of the surface proteins have been characterized biochemically. Focus on the external surface of trematodes has also been spurred by the belief that the tegument is the principal site of action of the important antischistosomal drug praziquantel.

3.1. External Surfaces 3.1.1. Structural considerations 3.1.1.1. Gross and microscopic anatomy. Like cestodes, trematodes are bounded by a layer of cytoplasm, undivided by internal membranes, termed a syncytial tegument (Fig. 12.2). In schistosomes the tegument is about 4 #m thick. It is limited externally by a heptalaminate membrane that consists of two closely apposed lipid bilayers. Based on the distribution of intramembranous particles observed in freeze-fracture studies, the inner bilayer is the true plasma membrane of the parasite. The membrane extends inward 1-2 #m along numerous pits that branch and interconnect to form a lattice which increases the surface area of the parasite at least 10-fold (28). The tegument of F. hepatica is much thicker and less pitted than that of schistosomes. It is bounded externally by a standard lipid bilayer from which extends a thin coat of glycoprotein

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FIG. 12.2 Schematicof the tegument and underlying tissues in Schistosoma mansoni. Abbreviations: hm, heptalaminate membrane; db, discoid body; mv, multilaminate vesicle; s, spine; si, surface invagination; bl, basal lamellum; cm, circular muscle; lm, longitudinal muscle; gj, gap junction; cc,cytoplasmic channel; pc, parenchymal cell; cy, cyton; n, nucleus. After ref. 27.

with projecting side chains of gangliosides and oligosaccharides (29). The function of this thin glycocalyx is unknown. The syncytial tegumental cytoplasm in schistosomes contains mitochondria and two major inclusions: discoid bodies and multilaminate vesicles. Discoid bodies are precursors of the dense ground substance of the cytoplasm as well as the surface bilayer (30). They may also break down to form crystalline spines which are most prominent on the dorsal surface of male schistosomes. Multilaminate vesicles consist of concentric whorls of membrane which migrate to the surface and form the outer bilayer. Mitochondria in the syncytial tegument are sparse, small and contain few cristae. Tegumental nuclei, most mitochondria and ribosomes are contained in subtegumental cells (cytons) located beneath the layers of muscle that underlie the syncytium. The subtegumental cells are connected to the syncytium through cytoplasmic channels that contain numerous microtubules. Inclusion bodies synthesized in the subtegumental cells arise from Golgi bodies and are transported to the syncytium through these channels. This process is interrupted by agents which interfere with protein synthesis or microtubule assembly (31). The tegumental surface membrane is constantly replaced by multilaminate vesicles that migrate to the apical region of the syncytium and fuse with the plasma membrane (32). The half-life of the outer membrane is about 3 hours (33).

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Much less is known about the synthesis and assembly of structural components in the tegument of F. hepatica.

3.1.1.2. Biochemistry and molecular biology of structural components. The schistosome tegument contains several phospholipids, the most abundant being phosphatidylcholine (57%), phosphatidylethanolamine (17%), sphingomyelin, lysophosphatidylcholine, phosphatidylinositol and cerebroside glycolipids (7, 34). Palmitic acid and oleic acid are the most abundant fatty acids (35). These molecules are derived from the host, as schistosomes are unable to synthesize cholesterol or long-chain fatty acids de novo (36). However, schistosomes can interconvert fatty acids and cleave the polar head from some phospholipids (37). The protein composition of the surface membrane of the tegument of schistosomes or F. hepatica has not been fully characterized. In schistosomes, this membrane possesses a range of enzymatic activities, including alkaline phosphatase, Ca 2 § and glycosyl transferase (38-40). In addition, ouabain binding sites on the surface of the tegument indicate the presence of Na§ (41). A large number of surface-associated proteins in schistosomes have been detected immunologically. Some are embedded in the tegumental membrane, whereas others are anchored by glycosylphosphatidylinositol. At least one is dependent on palmitoylation for membrane attachment (42). Unfortunately, functional information on these surface-associated proteins is quite limited. In some cases, nucleotide sequence analysis of genes that encode immunoreactive proteins has evolved biochemistry from antigenicity; paramyosin was found to be a partially protective tegumental antigen (43). These studies also showed that the surface spines are composed of paracrystalline arrays of actin filaments. Glutathione S-transferase was identified in similar experiments. Members of this enzyme family are present in the tegumental cytoplasm of schistosomes (44) and F. hepatica (45), presumably acting to protect the animals from oxidants. Interestingly, the enzyme is also found in the intestinal epithelium of F. hepatica (45). Carbohydrates exposed on the surface of schistosomes include mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. These sugars are linked to glycolipids and glycoproteins in the tegumental membrane, which also contains the enzymes required for their biosynthesis (46). Surface glycoproteins are anchored to the outer lipid bilayer by glycosylphosphatidylinositol through a process mediated by an endogenous phosphatidylinositol-specific lipase (47). 3.1.1.3. Developmental biology, synthesis and assembly. Schistosome cercariae undergo profound structural and biochemical transformation following penetration into the definitive host (48). During penetration, they lose their tails and begin to lose their fibrillar surface coat, or glycocalyx. Detachment of the glycocalyx follows secretion of proteases from the surface; these proteases may also play a role in tissue penetration (49, 50). By one hour after penetration, multilaminate vesicles begin to migrate to the outer surface of the tegument. During this period, numerous microvilli form from the outer membrane of the tegument, then are rapidly shed in a process that leads to the formation of the heptalaminate outer membrane (32). This protein shedding process may play an important role in the development of concomitant immunity, since it may prime the host to mount immune responses against subsequent challenge

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by cercariae(51). Formation of the heptalaminate membrane is complete within three hours after penetration. At this point, the tegument of the schistosomulum and adult stages are morphologically similar, as are their sterol and phospholipid compositions (52). Much higher levels of several surface proteins are expressed by immature stages than by adult schistosomes, and many surface proteins are differentially expressed on the surface of adult males and females (53). A Ca 2 +-binding protein is found in the tegument of developing larvae and may play a role in surface remodeling (54).

3.1.2. Functional biology 3.1.2.1. Biological functions of the structure. The trematode tegument is structurally adapted for transport, immune evasion and communication with the neuromuscular system (via gap junctions). Numerous pits at the surface of the tegument markedly increase the surface area of the parasite, which is consistent with a transport function (see below). The fact that all of the pits in schistosomes examined in situ contain erythrocytes (55) suggests that these structures are open to the external environment. The tegument is less pitted in F. hepatica, but numerous invaginations of the surface effectively increase the surface area. The double bilayer at the surface of the tegument in schistosomes is common only to blood-dwelling trematodes; species that inhabit other environments, including F. hepatica, are limited by a standard lipid bilayer (56). The double membrane in schistosomes may satisfy a structural requirement that allows rapid replacement of old or immunologically damaged membrane by new membrane derived from the multilaminate vesicles. It may also permit host antigens, largely blood group glycolipids and glycoproteins, to be incorporated into the surface membrane of the parasite, thereby disguising it from immune attack (57). Some lipid components of this membrane, such as lysophosphatidylcholine, may protect the organism by lysing leukocytes (58). Several structural components of the tegument suggest its involvement with the neuromuscular system. The tegument is electrically coupled by gap junctions to underlying muscle bundles in schistosornes (59), allowing changes in electrochemical gradients at the surface of the parasite to be transmitted directly to the muscle fibers below, presumably through gap junctions (60). The dorsal surface spines may serve as holdfasts to maintain the parasites within blood vessels or the bile duct. The tegument also contains acetylcholinesterase covalently anchored to the membrane through phosphatidylinositol (61). The surface location of this enzyme may destroy host acetylcholine in the circulation, protecting the parasite from its neuromuscular effects. 3.1.2.2. Nutrient absorption. A role for the tegument in nutrient absorption can be inferred on the basis of structural (above), biochemical and ecological considerations (62). Its numerous pits and channels provide an enormous surface area for absorption, and the tegument is constantly exposed to a nutrient-rich environment. Many enzymes that function in amino acid absorption are located in the tegument, and some of these, such as leucine aminopeptidase, are not present in the gut. Trematodes possess an incomplete digestive tract, and both S. mansoni and F. hepatica can survive extended in vitro incubations in the absence of detectable nutrient absorption across the intestine (63, 64). Glucose absorption in trematodes can be detected during immature stages of the life cycle, which lack an intestine (65).

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Adult schistosomes depend solely on host plasma glucose for energy. Physiological experiments suggested that glucose flux across the tegument occurs by an active Na +-dependent, carrier-mediated process (66, 67). The inward-directed Na + gradient that is maintained, in part, by Na+/K+-ATPase in the tegument, was thought to provide the driving force for this process. However, cloning and functional expression of schistosome glucose transporters in Xenopus laevis oocytes revealed that transport is Na +-independent and ouabain-insensitive (68). The discrepancy between physiological and molecular biological data may arise because manipulations of Na + in intact worms may lead to reductions in viability that are reflected in decreased glucose uptake. Alternatively, it is possible that Na+-dependent glucose transporters predominate in adult animals, but were not identified in the cloning experiments. Nevertheless, this example illustrates the power of recombinant DNA technology to spur progress in understanding the basic biology of parasites. Most glucose absorption occurs across the dorsal tegument in male worms; there is no evidence for regional differences in females (69). An intriguing aspect of glucose uptake by schistosomes is that, for worm pairs in copula(the normal condition in vivo), all or most glucose obtained by the female is supplied by the male partner (70). In addition, mated pairs absorb more glucose than unmated schistosomes, even though the total apparent surface area exposed to the external medium is much reduced. Increased glucose uptake in pairs may stem from increased rate or number of transport sites on the dorsal surface of the male (70). Amino acid uptake by trematodes occurs primarily across the tegument, as mechanical ligation of the pharynx or drug treatments that induce regurgitation do not significantly affect uptake kinetics. Most evidence suggests that amino acid absorption by F. hepatica occurs by passive diffusion, as uptake is linear over a wide range of concentrations and there is no evidence for competitive inhibition among related amino acids (71). In schistosomes, it appears that some amino acids, such as methionine, glutamate, arginine and alanine, are absorbed across the tegument by a combination of passive diffusion and a carrier-mediated system (72). The number of distinct amino acid uptake systems in schistosomes is unknown, though there is evidence for at least two and possibly three. These systems have a high affinity for amino acids only, as methionine uptake is not inhibited by other organic compounds, and moving the amino group to carbons adjacent to the ~-carbon reduces the affinity of the transport system (72). Trematodes do not synthesize cholesterol de novo, but acquire it from the host. Uptake occurs via the tegument, primarily at the dorsal surface of males (73), and the cholesterol is then redistributed throughout the body (74). Cholesterol, like glucose, is transferred between partners in copula (75). Other lipids are also transported across the tegument, and regionally specified areas of this surface may be involved (74). 3.1.2.3. Water balance. Like cestodes, trematodes have a limited capacity to regulate the osmolarity of their body fluids. During the process of transformation from the cercarial stage to the schistosomulum stage following penetration of the host organism, schistosomes become much more permeable to water. When placed in hypo-osmotic medium, adult schistosomes (and other trematodes) gain weight rapidly, then exhibit a slow recovery to control weight levels. This response indicates a capacity for osmoregulation, and is interpreted as an initial osmotic uptake of water across the tegument

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followed by a slower efflux of ions from the tissue, with water following (76). In hyperosmotic medium, schistosomes slowly lose weight due to osmotic efflux of water (77). How trematodes re-establish osmotic equilibrium is not known. They have a well-developed protonephridial system that may function in osmoregulation. However, there is no evidence that the protonephridial system responds to ionic or osmotic stress (78). Osmoregulation could also be mediated by active transport of ions across the tegument. In S. mansoni, the Na § concentration in extracellular fluid is approximately equal to that in the bathing medium over a wide range. The Na § concentration in the tegumental syncytium, however, is maintained at a constant level that is much lower than that in the extracellular fluid or the external medium (76).

3.1.2.4. Transport of inorganic ions. In both S. mansoni and F. hepatica, the low Na + concentration in the tegument is maintained by an Na+/K+-ATPase (27,41). This enzyme contributes extensively to the maintenance of an electrochemical gradient across the outer tegumental membrane which, in S. mansoni, results in a membrane potential of - 6 0 mV (79). As indicated by the effect of changing external ion concentrations on tegumental membrane potential, the tegument is more permeable to K + than to Na + or C1-. This is a common feature of transporting epithelia in a wide range of vertebrates and invertebrates. In addition, the schistosome tegument contains at least one type of voltage-dependent ion channel (80). This channel has been studied using inside-out patches of outer tegumental membrane from adult female S. mansoni. It is cationselective (i.e., conducts Na + and K +, but not C1-) and exhibits a large unitary conductance. 3.1.2.5. Excretion. Trematodes excrete a number of metabolic end-products, including lactate, amino acids, NH,~ and H +. Protonephridial organs and the tegument probably play a role in excretion. Levels of Na + within the tegumental syncytium become elevated after acidification of the external medium. Amiloride and low Na + medium interfere with recovery from an acid load, suggesting the existence of an Na +-H + exchanger in the tegument (81).

3.2. Internal Surfaces 3.2.1. Structural considerations 3.2.1.1. Gross and microscopic anatomy. The digestive tract of trematodes consists of a mouth, pharynx and esophagus that branches to form a pair of intestinal caeca, which extend posteriorly and end blindly. The caeca consist of a single layer of epithelial cells supported by a thin layer of longitudinal and circular muscle fibers. The epithelial layer is syncytial in schistosomes, but is cellular in F. hepatica (27). The surface area of the caeca is amplified up to 100-fold by numerous digitiform microvilli or pleomorphic lamellae. The gut epithelial cells contain numerous mitochondria and ribosomes, and most of the cytoplasm is occupied by endoplasmic reticulum, Golgi bodies and secretory bodies.

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3.2.1.2. Developmental biology. In schistosomes, the gut becomes functional within a few hours after penetration of the definitive host. F. hepatica begin to ingest host tissue immediately upon excystment of the metacercaria, with breakdown products of host tissue apparent in the caeca within a few hours. In newly excysted F. hepatica, the intestinal caeca appear to serve as reservoirs for enzymes used to penetrate the wall of the host gut during migration to the liver. Upon reaching the liver, the immature flukes begin to feed on hepatic cells, and the caeca are transformed into digestive/absorptive organs (46). Adult F. hepatica ingest mainly erythrocytes, but also other host tissues, including bile duct epithelium and mucus (27). Digestive enzyme activities associated with the gastrodermis of adult F. hepatica include those already listed for schistosomes plus amino peptidase and lipase (82) and a cathepsin L-like protease (83). Little is known about the biochemistry associated with development of the trematode gastrodermis. Erasmus (84) gives a detailed review of the functional morphology of the gastrodermis in larval and adult trematodes. 3.2.2. Functional biology 3.2.2.1. Nutrient digestion and absorption. The importance of the gut surface in trematodes as a site for nutrient digestion and absorption is suggested on the basis of several observations. Schistosomes begin to feed on host blood within 4 - 6 hours after penetration. In vitro, immature stages can survive several days without erythrocytes, but their growth and development is severely retarded (85). Females ingest many more erythrocytes than males, presumably in response to the demand for amino acids and proteins used in egg formation (27). Lysis and digestion of erythrocytes begins in the esophagus of schistosomes, and appears to be initiated by the release of dense secretory bodies from gland cells in the posterior esophagus (86). A wide range of enzymes involved in digestion and processing of nutrients are found in gut epithelial cells of trematodes, including proteases, esterases, acid phosphatases and glucose-6-phosphatase (27). The most thoroughly studied is an abundant hemoglobinase present in gut epithelial cells of S. mansoni (Chapter 5). It degrades hemoglobin released from ingested erythrocytes and is a 31 kDa acid thiol protease similar to cathepsin B or L. The gene that encodes this enzyme has been cloned (87). Many other peptidases are also present in the schistosome gut (49), but their specific roles in digestion have not been fully elucidated. A cathepsin L-like protease has also been found in vesicles in the gut epithelia of F. hepatica (83). The trematode gastrodermis epithelium is probably the principal site of absorption for large-molecular-weight nutrients. These nutrients seem to be critical for growth and long-term survival and for egg production by females (88a). The mechanisms of absorption are uncharacterized. Endocytosis occurs to a very limited extent in F. hepatica, but there is no evidence for this process in schistosomes. There is no information available on the role of the trematode gastrodermis in the transport of inorganic ions or water. 3.2.2.2. Excretion. There is little information on the role of the trematode gastrodermis in excretion of metabolic end-products. Most excretion is believed to occur by way of the highly developed protonephridial system, which is distinct from the tegument or the gastrodermis. In schistosomes and F. hepatica, the byproducts of erythrocyte

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digestion collect in the gastrodermis and are periodically regurgitated. In schistosomes, cellular organelles and macromolecules derived from the gastrodermis are constantly discharged across the internal surface into the lumen (86). This process has not been studied in F. hepatica.

4. NEMATODES The rigid cuticle that is the external surface of nematodes is quite distinct from the tegument of cestodes and trematodes. Nematodes also possess a functional gut that ends in a muscularly controlled rectum. Although the various niches occupied by nematodes have led to evolutionary surface adaptations particularly suited for specific environments, the general features of their surface biology are well conserved. More is known about the biochemistry of nematode surfaces than about the surfaces of other helminths, for several reasons. Parasitic nematodes pose a considerable challenge to veterinary and human medicine, and attention has been focused on them for economic reasons. The size of Ascaris suum has greatly facilitated physiological and biochemical studies. Finally, a great deal of work has been performed on the free-living nematode Caenorhabditis eleoans. The power of genetics has illuminated a large number of basic biological and biochemical phenomena in this organism. Fortunately, A. suum and C. eleoans provide data that can generally be extrapolated to other nematodes. Many reviews on the surfaces of nematodes are available (13, 88a-101).

4.1. External surfaces 4.1.1. Structural considerations 4.1.1.1. Gross and microscopic anatomy. Nematodes are bounded externally by a complex, multilayered cuticle that extends into and lines the pharynx, rectum, cloaca and other orifices. The cuticle represents an extracellular secretion of the hypodermis, an anatomical syncytium that forms a continuous cellular layer immediately beneath the cuticle and is specialized for transport and secretion. Though the cuticle and hypodermis are morphologically distinct, they represent a functional unit (Fig. 12.3). Nematode cuticles are tremendously diverse in structure. Among species that parasitize vertebrates, the cuticle of the adult stage typically consists of four distinct layers: the epicuticle, cortex, medial layer and composite basal layer. The outermost layer, or epicuticle, is 6-30 nm thick and appears trilaminate in electron micrographs. Considerable controversy exists over whether this layer should be considered the limiting 'membrane' of nematodes (29,93,98). The epicuticle is not dissociated by treatments that typically dissolve membranes (99) and lipophilic markers do not exhibit the lateral mobility characteristic of membranes when inserted into the surface of nematodes (100a). Furthermore, the structure is devoid of intramembranous particles that are normally associated with cell membranes (100b). At least some stages and species of nematodes also exhibit a loosely associated surface coat. This coat is secreted through the excretory pore and from the esophagous (89).

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